Multi-valent human immunodeficiency virus antigen binding molecules and uses thereof

ABSTRACT

This disclosure provides a multimeric human immunodeficiency virus (HIV) protein binding molecule, e.g., an dimeric IgA or a pentameric or hexameric IgM binding molecule, comprising at least two bivalent binding units, or variants or fragments thereof, each comprising at least two antibody heavy chain constant regions or fragments thereof, wherein each heavy chain constant region or fragment thereof is associated with an HIV antigen binding domain. Also provided are compositions comprising the multimeric binding molecules, polynucleotides encoding the multimeric binding molecules, and methods to make and use the multimeric binding molecules.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/566,485, filed Oct. 13, 2017, which is US National Stage Entry of PCTApplication No. PCT/US2016/027979, filed Apr. 15, 2016, which claims thebenefit of U.S. Provisional Application Ser. No. 62/149,460, filed onApr. 17, 2015, which are hereby incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 11, 2017, isnamed 57912-170015_SL.txt and is 196589 bytes in size.

BACKGROUND

Human immunodeficiency virus (HIV) is a retrovirus of the Lentivirusfamily. HIV is a single stranded, positive sense enveloped RNA virus.HIV causes acquired immunodeficiency syndrome (AIDS) which leads tofailure of the immune system due to destruction of T cells, macrophagesand dendritic cells. HIV has a very high rate of genetic variability.Two types of HIV have been identified, including HIV-1 and HIV-2, bothof which are transmitted by sexual contact or through blood and bothcause AIDS. The two viruses differ in that HIV-1 is more prevalent, morevirulent, and more easily transmitted than HIV-2. HIV-1 can be furtherdivided into three groups based on sequence differences in the envelope(env) gene, group M, group N group O, and group P. Group M of HIV-1 isfurther divided into at least nine subtypes (or clades) based ondifference in genomic sequence and geographic distribution (subtypes A,B, C, D, F, G, H, J and K). The structure of the HIV RNA genome includesnine genes: gag, pol, env, tat, rev, nef, vif, vpr, and vpu. Some HIVgenomes include a tenth gene called tev, a fusion of tat, env, and rev.These genes encode the following proteins:

Viral Structural Proteins

-   gag proteolytically processed to yield matrix protein (p17, MA),    capsid protein (p24, CA), nucleocapsid protein (p7, NC), spacer    peptide 2 (SP2, p1) and P6 protein-   pol proteolytically processed to yield reverse transcriptase (RT),    RNase H, integrase (IN) and HIV protease (PR)-   env gp160 envelope protein; processed proteolytically to yield gp120

Essential Regulatory Elements

-   tat RNA binding transcriptional activator protein-   rev Rev protein, which is a sequence-specific RNA binding regulator    protein

Accessory Regulatory Proteins

-   vpr Vpr is lentivirus nucleocytoplasmic shuttling/transport    regulatory protein-   vif Vif is a cell-specific regulatory phosphoprotein-   nef Nef is an N-terminal myristoylated membrane-associated    regulatory phosphoprotein-   vpu Vpu is an HIV-1 specific integral membrane regulatory    phosphoprotein-   tev tev is a tat/env/rev fusion gene yielding a fusion protein

The HIV envelope protein, gp160 is proteolytically cleaved by a hostcell protease to yield gp120 and gp41. These two molecules form a capand stem structure protruding from the viral envelope, also referred toas the spike. The cap is composed of three copies of gp120 and the stemis composed of three copies of gp41. The stem anchors the gp120/gp41complex to the viral envelope. The envelope glycoprotein gp120 isexpressed both on the surface of infected cells and the viral envelopeof viral particles. The gp120 portion of the Env protein is responsiblefor binding to the CD4 receptor on target cells, such as helper T cells,enabling the virus to fuse to the host cell. Structure of the HIV spikeglycoprotein is shown in FIG. 1.

The viral protein gp120 has been intensely focused upon in vaccinedevelopment research since it is the main point of contact and entryinto host cells. However, the mechanism of HIV entry into cells includesmasking of gp120 epitopes by covalently attached sugar moieties. It isonly when the HIV virion is in close proximity to a host cell that theportion of gp120 that interacts with host cell receptors is unmasked.Likewise, glycoprotein gp41 is non-covalently associated with gp120 andbecomes exposed only after gp120 binds to its target and undergoes aconformational change. The conformational change triggered by binding tothe host cell allows gp41 to assist in the fusion of the virion to thehost cell. Thus, gp41 has also received the attention of clinicalresearch as a potential target for antiviral drugs. While gp120 and gp41are highly immunogenic, the proteins can vary substantially between HIVtypes, groups, and/or clades.

Moreover, the proteins frequently mutate to form antigenic variants andrapidly evolve to evade the host immune response.

Accordingly, the development of therapeutic monoclonal antibodies thatcan cross react with antigenic determinants on a large number of HIVtypes, groups, and clades is currently an area of intense investigation.The broadly neutralizing HIV antibodies, or bnAbs, are described in theliterature and have been collected at the web site “Broadly NeutralizingAntibodies Electronic Resource,” www.bnaber.org (Eroshkin A M, et al.,Nucleic Acids Res. 42(1):D1133-9 (2014)).

The epitopes of the bnAbs are found on the heterotrimer HIV envelopespike, composed of gp41 and gp120 and the surrounding glycan layer. Abroad range of spike epitopes, both linear and conformational, have beenidentified, and four epitopes bound by bnAbs have been extensivelycharacterized: the membrane proximal external region (MPER), the CD4binding site, the variable region 1/variable region 2 (V1/V2) loop andthe variable region 3 (V3) loop (Hepler N L. et al. PLOS Comp. Biol.10(9): e1003842 (2014)).

MPER is located on the gp41 protein in the base of the HIV spike(Montero M. et al. MMBR 72(1):54-84 (2008)). BnAbs interacting with theCD4 binding site on gp120 can mimic CD4 binding. For example, thebinding of bnAb VCR01 to the CD4 binding site causes a conformationalshift that is thought to disable the virus receptor thereby neutralizingthe HIV virus (Scheid J F. et al., Science 333(6049):1633-7 (2011)). TheV1/V2 loop on gp120 has been shown to be one of the most frequentepitopes for potent bnAbs (Moore P. L. et al. J. Virol. 69(9):5723-33(2011)). The V3 loop on gp120 has been targeted by neutralizingantibodies in a quaternary-structure specific manner. The glycan shieldcan also contribute to the conformation of neutralizing HIV antibodyepitopes and make direct contacts with bnAbs; conversely the glycans caninhibit antibody binding through steric hindrance.

HIV is thought to remain dormant in reservoir cells in various tissuesthroughout the body due to the immunoprivileged status of certaintissues, such as the central nervous system, the genitourinary tract,and lymphoid organs. (See, Iglesias-Ussel et al., AIDS Rev., 13:13-29,2011). It has been postulated that the gastrointestinal tract (GIT) is aprimary target for HIV infection, and a major cellular reservoir due tothe abundance of macrophages located at mucosal sites in the GIT. (See,Brown et al., Clin. Vacc. Immunol., 21(11):1469-1473, 2014). However,latently infected memory T cells are the largest and best understoodreservoir for HIV. Even when treated with Highly Active AntiretroviralTherapy (HAART), the T cell reservoir alone has a remarkably longhalf-life (Finzi, D., et al. 1999. Nature Med 5:512-7) resulting inrapid rebound and virus reemergence upon cessation of therapy. It isbelieved that infection of some CD4+ T cells can be followed bytransition of the infected T cell into a quiescent state and ultimatelyformation of a memory CD4+ T cell which contains an integrated genomiccopy of the viral genome (proviral DNA) which is not expressed until alater time when transcription is triggered. Others believe that suchreservoir cells are not be truly silent, but instead persistently orstochastically produce small amounts of virus. Memory T cells are idealHIV reservoir cells since they are quiescent and do not undergo celldivision, differentiation, or activation and their transcriptionalmachinery therefor exhibits only minimal activity. However, uponactivation, memory T cells can produce large quantities of virus.Targeting of dormant HIV reservoirs is a subject of intensive study andrepresents the next major hurdle in managing and ultimately eliminatingHIV infection. These HIV-infected reservoir cells are most ofteninfected with mutated variants of the originally-infecting HIV virus.(See, Picker et al., Nature, 517:381-385, 2015).

Thus, chronically infected individuals, although not harboringdetectable levels of expressed HIV nonetheless continue to carry HIV andthe potential to succumb to HIV infection, or infect others, withoutfurther exposure to HIV. There is presently no publically availabletreatment known to clear chronic HIV infection. Available therapiesmerely halt further infection by precluding the virus from replicating.Various monoclonal antibodies and combination therapies have beeninvestigated for the purpose of treating HIV, including chronic HIVinfection, but none have been commercialized. Therefore, there remains acontinuing need to develop new therapies targeting HIV, e.g., reservoircells harboring dormant HIV in chronic HIV infection. Thus, there is astrong need for more potent treatments that are readily available and donot present cost barriers to clinical application and availability.

SUMMARY

Disclosed are various embodiments of multimeric binding molecules thatpossess specificity for binding one or more HIV antigens, e.g.,gp120/gp41 antigens.

This disclosure provides a multimeric binding molecule that includes atleast two bivalent binding units, or variants or fragments thereof;where each binding unit includes at least two antibody heavy chainconstant regions or fragments thereof, where each heavy chain constantregion or fragment thereof is associated with an antigen binding domain,where at least one antigen binding domain specifically binds to a humanimmunodeficiency virus (HIV) antigen expressed on the surface of viralparticles, on the surface of HIV-infected cells, or a combinationthereof, and where the binding molecule is more potent in preventing,controlling or treating HIV infection than a corresponding referencesingle binding unit molecule including the HIV antigen binding domain.The corresponding reference single binding unit molecule can be, e.g.,an IgG antibody.

In certain aspects, the at least one antigen binding domain specificallybinds to the HIV spike protein, e.g., to an epitope on gp120, gp41, or acombination thereof. In certain aspects the epitope is situated in theimmunodominant region of gp41, the MPER, the CD4 binding site, the V1/V2loop, the V3 loop, the carbohydrates associated with these regions, or acombination thereof.

In certain aspects, the provided binding molecule is multispecific,e.g., bispecific, including at least two non-identical antigen bindingdomains. The two non-identical antigen binding domains can specificallybind, without limitation, to different epitopes of a common HIV antigen,to different HIV antigens, or to an HIV antigen and a heterologousantigen.

In certain aspects, the provided binding molecule is a dimeric bindingmolecule that includes two bivalent IgA binding units or fragmentsthereof and a J-chain or fragment thereof or variant thereof. Accordingto these aspects, each binding unit can include two IgA heavy chainconstant regions or fragments thereof each associated with an antigenbinding domain. In certain aspects a dimeric binding molecule asprovided herein can further include an associated secretory component,or fragment or variant thereof. A dimeric binding molecule as providedherein can include the Cα2 domain and/or the Cα3-tp domain if the IgAconstant region, and can in some aspects further include the Cα1 domain.In certain aspects the IgA heavy chain constant region is a human IgAheavy chain constant region. An IgA-based dimeric binding molecule asprovided herein can include, in some aspects, two IgA heavy chains eachincluding a VH situated amino terminal to the IgA constant region orfragment thereof, and two immunoglobulin light chains each including aVL situated amino terminal to an immunoglobulin light chain constantregion.

In certain aspects the provided binding molecule is a pentameric or ahexameric binding molecule including five or six bivalent IgM bindingunits, respectively, where each binding unit includes two IgA heavychain constant regions or fragments thereof each associated with anantigen binding domain. In certain aspects the IgM heavy chain constantregions or fragments thereof can each include a Cμ3 domain and a Cμ4-tpdomain and can in some aspects, further include a Cμ2 domain, a Cμ1domain, or any combination thereof. Where the provided binding moleculeis pentameric, it can further include a J-chain, or fragment thereof, orvariant thereof. In certain aspects, the IgM heavy chain constant regionis a human IgM constant region.

In certain aspects, the disclosure provides a bi- or multispecificdimeric or pentameric binding molecule that includes a J-chain. TheJ-chain can be a modified J-chain including, e.g., a binding domain. Incertain aspects the modified J-chain is derived from a human J-chain,and can include the amino acid sequence 23 to 159 of SEQ ID NO: 2, or afunctional fragment thereof. In certain aspects the binding domain is apolypeptide sequence fused in frame with the J-chain or fragmentthereof, either with or without a peptide linker. In certain aspects thepeptide linker can include at least 5 amino acids, but no more than 25amino acids. In certain aspects the peptide can consist of SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104. In certainaspects the modified J-chain can include the formula X[L_(n)]J orJ[L_(n)]X, where J includes a mature native J-chain or functionalfragment thereof, X includes a heterologous binding domain, and [L_(n)]is a linker sequence consisting of n amino acids, where n is a positiveinteger from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5,10, 15, or 20.

In certain aspects the binding domain is situated at the C-terminus ofthe J-chain or fragment thereof. In certain aspects the binding domainis situated at the N-terminus of the J-chain or fragment thereof. Incertain aspects the binding domain is inserted within the J-chain orfragment thereof. In certain aspects the binding domain of the modifiedJ-chain is an antibody or antigen binding fragment thereof, e.g., anF(ab′)₂, an F(ab)₂, an Fab′, an Fab, an Fv, an scFv, or a single domainantibody, e.g., a VHH. In certain aspects, the binding domain of themodified J-chain binds to one or more effector cells, e.g., T-cells,natural killer (NK) cells, macrophages and/or neutrophils. Where theeffector cell is a T-cell, the binding domain can bind, e.g., to CD3 orCD8 on the T-cell. Where the effector cell is an NK cells, the bindingdomain can bind, e.g., to one or more of CD16, CD64, and/or NKG2D on theNK cell. Where the effector cell is a macrophage, the binding domain canbind to, e.g., CD14 on the macrophage. Where the effector cell is aneutrophil, the binding domain can bind to, e.g., CD16b and/or CD177 onthe neutrophil. In certain aspects, the heterologous polypeptideincludes SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or acombination thereof. In certain aspects the modified J-chain includesSEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or acombination thereof. In certain aspects the modified J-chain can furtherinclude a signal peptide.

In certain aspects, each binding unit of a binding molecule as providedherein can include two heavy chains each including a VH situated aminoterminal to the constant region or fragment thereof, and twoimmunoglobulin light chains each including a VL situated amino terminalto an immunoglobulin light chain constant region. In certain aspects, atleast one binding unit includes two antigen binding domains thatspecifically bind to an HIV antigen expressed on the surface of viralparticles, on the surface of HIV-infected cells, or a combinationthereof. In some aspects the two heavy chains within the binding unitcan be identical. In some aspects the two light chains within thebinding unit are identical. The two light chain constant regions can be,e.g., human lambda constant regions or human kappa constant regions.

In certain aspects the binding molecule as provided herein can includeat least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, at least eleven, ortwelve antigen binding domains that specifically bind to an HIV antigenexpressed on the surface of viral particles, on the surface ofHIV-infected cells, or a combination thereof. In certain aspects atleast two, at least three, at least four, at least five, or at least sixof the binding units are identical. In certain aspects at least oneantigen binding domain of the binding molecule as provided herein canspecifically bind to an HIV spike protein expressed or presented on thesurface of HIV-infected reservoir cells, e.g., a cell in which HIVantigens are expressed at a low level compared to HIV-infected cells.

In certain aspects at least one antigen binding domain of the bindingmolecule as provided herein includes an antibody heavy chain variableregion (VH) and an antibody light chain variable region (VL), where theVH and VL can include the HCDR1, HCDR2, and HCDR3 regions, or HCDR1,HCDR2, and HCDR3 regions containing one or two single amino acidsubstitutions, and the LCDR1, LCDR2, and LCDR3 regions, or LCDR1, LCDR2,and LCDR3 containing one or two single amino acid substitutions, of theVH and VL amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ IDNO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 andSEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ IDNO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28,SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ IDNO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 andSEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ IDNO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO:50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54,SEQ ID NO: 55 and SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ IDNO: 59 and SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63and SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 andSEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ IDNO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 6,SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ IDNO: 81 and SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85and SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 andSEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ IDNO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO:98, or SEQ ID NO: 99 and SEQ ID NO: 100, respectively.

In certain aspects, at least one antigen binding domain of the bindingmolecule as provided herein includes an antibody heavy chain variableregion (VH) and an antibody light chain variable region (VL), where theVH and VL include, respectively, amino acid sequences that are at least80%, at least 85%, at least 90%, at least 95% or 100% identical to aminoacid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ IDNO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12,SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ IDNO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 andSEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ IDNO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO:34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38,SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 andSEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 55 and SEQ IDNO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO:60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64,SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ IDNO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 6, SEQ ID NO: 77 and SEQID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO:82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86,SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ IDNO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, SEQ ID NO: 95and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, or SEQ ID NO: 99 andSEQ ID NO: 100.

In certain aspects the binding molecule as provided herein is morepotent in preventing, controlling or treating HIV infection, enhancingviral clearance, controlling HIV infectivity, and/or controlling HIVgrowth than a corresponding reference single binding unit moleculeincluding the HIV-binding antigen binding domain. In certain aspects thebinding molecule can be more potent in neutralizing HIV, can bind to andneutralize more diverse HIV variants or clades, or a combinationthereof, than a corresponding reference single binding unit moleculethat includes the HIV-binding antigen binding domain. In certain aspectsthe binding molecule can effect more potent antibody mediated,complement mediated, or cell mediated, e.g., T-cell mediated, killing ofHIV infected cells than a corresponding reference single binding unitmolecule that includes the HIV-binding antigen binding domain. Incertain aspects the binding molecule can provide equivalent benefit at alower dosage than a corresponding reference single binding unit moleculethat includes the HIV-binding antigen binding domain.

The disclosure further provides an isolated IgM antibody or fragmentthereof that includes a J-chain, or functional fragment or variantthereof, and five binding units, each including two heavy chains and twolight chains, where each heavy chain or fragment thereof includes ahuman Mu constant region or fragment thereof, and the heavy chainvariable region amino acid sequence SEQ ID NO: 7, and where each lightchain includes a human kappa constant region and the light chainvariable region amino acid sequence SEQ ID NO: 8; where the antibody orfragment thereof can assemble into a pentameric IgM antibody that canspecifically bind to the CD4 binding site of the HIV spike glycoprotein.In certain aspects the IgM antibody or fragment thereof can include theheavy chain amino acid sequence SEQ ID NO: 113 and the light chain aminoacid sequence SEQ ID NO: 114. In certain aspects the J-chain can includeamino acids 23 to 159 of the amino acid sequence SEQ ID NO: 2 or afunctional fragment thereof. In certain aspects the J-chain or fragmentthereof can be a modified J-chain that further includes a heterologouspolypeptide that can be directly or indirectly fused to the J-chain. Incertain aspects the heterologous polypeptide can be fused to the J-chainor fragment thereof via a peptide linker, e.g., a peptide linker thatincludes at least 5 amino acids, but no more than 25 amino acids, e.g.,a peptide linker consisting of SEQ ID NO: 101, SEQ ID NO: 102, SEQ IDNO: 103, or SEQ ID NO: 104. In certain aspects the heterologouspolypeptide can be fused to the N-terminus of the J-chain or fragmentthereof, the C-terminus of the J-chain or fragment thereof, or to boththe N-terminus and C-terminus of the J-chain or fragment thereof. Incertain aspects the heterologous polypeptide can include a bindingdomain, e.g., the heterologous polypeptide can be an antibody or antigenbinding fragment thereof, e.g., a scFv fragment, e.g., a scFv fragmentthat can specifically bind to CD3. In certain aspects the modifiedJ-chain can include a heterologous polypeptide that includes the aminoacid sequence SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ IDNO: 111. In certain aspects the modified J-chain can further include asignal peptide.

The disclosure further provides a composition that includes the bindingmolecule or the IgM antibody provided herein.

In addition, the disclosure provides a polynucleotide that includes anucleic acid sequence encoding a polypeptide subunit of the bindingmolecule as provided herein, where the polypeptide subunit includes theIgM heavy chain constant region and at least the antibody VH portion ofan antibody binding domain that specifically binds to an HIV spikeprotein antigen expressed on the surface of viral particles, on thesurface if HIV-infected cells, or a combination thereof. In certainaspects the polypeptide subunit can include a human IgM constant regionor fragment thereof fused to the C-terminal end of a VH domain thatincludes: the HCDR1, HCDR2, and HCDR3 domains, or the HCDR1, HCDR2, andHCDR3 domains containing one or two single amino acid substitutions inone or more HCDRs, of the VH amino acid sequence SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO:83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ IDNO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99; or an amino acidsequence at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to the SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ IDNO: 97, or SEQ ID NO: 99.

In addition, the disclosure provides a polynucleotide that includes anucleic acid sequence encoding a polypeptide subunit of the bindingmolecule as provided herein, where the polypeptide subunit includes theantibody VL portion of an antibody binding domain that specificallybinds to an HIV spike protein antigen expressed on the surface of viralparticles, on the surface of HIV-infected cells, or a combinationthereof. In certain aspects, the polypeptide subunit includes a humanantibody light chain constant region or fragment thereof fused to theC-terminal end of a VL that includes: the LCDR1, LCDR2, and LCDR3domains, or the LCDR1, LCDR2, and LCDR3 domains containing one or twosingle amino acid substitutions in one or more LCDRs, of the VL aminoacid sequence SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 6, SEQ ID NO: 78, SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO:98, or SEQ ID NO: 100; or an amino acid sequence at least 80%, at least85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ IDNO: 74, SEQ ID NO: 6, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92,SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100.

The disclosure further provides a composition that includes VH andVL-containing polynucleotide as provided herein. In certain aspects thepolynucleotides are situated on the same vector. In certain aspects thepolynucleotides are situated on separate vectors. In certain aspects thecomposition further includes a polynucleotide that includes a nucleicacid sequence encoding a J-chain, a modified J-chain, fragment thereof,or a variant thereof. In certain aspects, the polynucleotides aresituated on at least two separate vectors. In certain aspects, thepolynucleotides are situated on the same vector. The disclosure furtherprovides the vector or vectors as described, a host cell that includesthe provided polynucleotide or the provided polynucleotide composition,or the provided vector or vectors. The disclosure further provides amethod of producing the binding molecule provided herein, where themethod includes culturing the provided host cell, and recovering thebinding molecule.

The disclosure further provides a method of preventing, controlling, ortreating HIV infection, or controlling human immunodeficiency virus(HIV) infectivity, where the method includes contacting a mixture of HIVand HIV-susceptible cells with the binding molecule provided herein;where the binding molecule is more potent in preventing, controlling ortreating HIV infection, or in controlling HIV infectivity, than acorresponding reference single binding unit molecule including theHIV-binding antigen binding domain. In certain aspects, the bindingmolecule exhibits increased potency in neutralizing HIV in infectedcells, as compared with the single binding unit molecule.

The disclosure further provides a method of treating an humanimmunodeficiency virus (HIV) infection in a patient, includingadministering to a patient infected with HIV the binding molecule asprovided herein; where the binding molecule is stronger, more potent inpreventing, controlling or treating HIV infection or controlling HIVinfectivity, or requires a lower binding molecule dose than acorresponding reference single binding unit molecule including theHIV-binding antigen binding domain. According to this method, thebinding molecule can exhibit increased potency in (i) reducing theinfectivity of an HIV virion, (ii) reducing the number of HIV-infectedcells, (iii) preventing HIV infection, (iv) enhancing viral clearance,(v) improving the signs and symptoms of HIV infection, or (vi) anycombination thereof, as compared with the corresponding reference singlebinding unit molecule. In certain aspects, the corresponding referencesingle binding unit molecule is an IgG antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Illustration of various antibody binding sites on the HIV spikeglycoprotein (Modified from Modified from Klein F., et al. Science341:1199-1204 (2013)).

FIG. 2A: Expression and assembly of the HIV02 antibodies, as measured bynon-reducing SDS native-PAGE.

FIG. 2B: Assembly of HIV12, HIV32 and HIV72 IgM+J proteins. Proteinswere electrophoresed in non-reducing SDS native-PAGE, transferred tomembrane and probed with anti-J antibody. Lane 1, reference IgM+J; lane2, HIV02; lane 3, HIV32; lane 4, HIV72.

FIG. 2C: Assembly of HIV72 IgG, IgM+J and the IgM+V5J bispecific. Lane1, Native markers, lane 2, HIV72 IgG; lane 3, HIV72 IgM; lane 4, HIV72IgM+J; lane 5 HIV72 IgM+V5J. The left side of the figure shows Coomassiestaining of the gel, and the right side shows an anti-J chain westernblot.

FIG. 3A-F: Effect of antigen coating concentration on the binding ofHIV02 IgG and HIV02 IgM+J to gp120 by ELISA. FIG. 3A: 1 μg/ml antigencoating; FIG. 3B: 0.8 μg/ml antigen coating; FIG. 3C: 0.6 μg/ml antigencoating; FIG. 3D: 0.4 μg/ml antigen coating; FIG. 3E: 0.2 μg/ml antigencoating; FIG. 3F: 0.1 μg/ml antigen coating.

FIG. 4: Comparison of HIV02 IgG and HIV02 IgM+J binding to gp120 byELISA. The IgM+J molecule is 20× better than the IgG molecule at lowantigen densities.

FIG. 5A-B: HIV02 IgG (FIG. 5A) and IgM+J (FIG. 5B) binding togp140-expressing CHO-PI cells.

FIG. 6: Neutralization of HIV isolates from multiple clades by HIV02IgM+J.

FIG. 7: Antigen-dependent T-cell activation by HIV02 IgM+V10J.

FIG. 8: PAGE analysis of HIV02+V10J HPLC-SEC column fractions.

FIG. 9: Antigen-dependent T-cell activation by SEC-purified HIV02IgM+V10J.

DETAILED DESCRIPTION Definitions

The term “a” or “an” entity refers to one or more of that entity; forexample, “a binding molecule,” is understood to represent one or morebinding molecules. As such, the terms “a” (or “an”), “one or more,” and“at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “non-naturally occurring” substance,composition, entity, and/or any combination of substances, compositions,or entities, or any grammatical variants thereof, is a conditional termthat explicitly excludes, but only excludes, those forms of thesubstance, composition, entity, and/or any combination of substances,compositions, or entities that are well-understood by persons ofordinary skill in the art as being “naturally-occurring,” or that are,or might be at any time, determined or interpreted by a judge or anadministrative or judicial body to be, “naturally-occurring.”

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation, andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a biological source or produced byrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide as disclosed herein can be of a size of about 3 or more, 5or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid, e.g., a serine or an asparagine.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated as disclosed herein, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the term “non-naturally occurring” polypeptide, or anygrammatical variants thereof, is a conditional term that explicitlyexcludes, but only excludes, those forms of the polypeptide that arewell-understood by persons of ordinary skill in the art as being“naturally-occurring,” or that are, or might be at any time, determinedor interpreted by a judge or an administrative or judicial body to be,“naturally-occurring.”

Other polypeptides disclosed herein are fragments, derivatives, analogs,or variants of the foregoing polypeptides, and any combination thereof.The terms “fragment,” “variant,” “derivative” and “analog” as disclosedherein include any polypeptides that retain at least some of theproperties of the corresponding native antibody or polypeptide, forexample, specifically binding to an antigen. Fragments of polypeptidesinclude, for example, proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of, e.g., a polypeptide include fragments asdescribed above, and also polypeptides with altered amino acid sequencesdue to amino acid substitutions, deletions, or insertions. In certainaspects, variants can be non-naturally occurring. Non-naturallyoccurring variants can be produced using art-known mutagenesistechniques. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or additions.Derivatives are polypeptides that have been altered so as to exhibitadditional features not found on the original polypeptide. Examplesinclude fusion proteins. Variant polypeptides can also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide can also refer to a subject polypeptide having one or moreamino acids chemically derivatized by reaction of a functional sidegroup. Also included as “derivatives” are those peptides that containone or more derivatives of the twenty standard amino acids. For example,4-hydroxyproline can be substituted for proline; 5-hydroxylysine can besubstituted for lysine; 3-methylhistidine can be substituted forhistidine; homoserine can be substituted for serine; and ornithine canbe substituted for lysine.

A “conservative amino acid substitution” is one in which one amino acidis replaced with another amino acid having a similar side chain.Families of amino acids having similar side chains have been defined inthe art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the present disclosure do not abrogatethe binding of the polypeptide or antibody containing the amino acidsequence, to the antigen to which the binding molecule binds. Methods ofidentifying nucleotide and amino acid conservative substitutions that donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32:1180-1 187 (1993); Kobayashi et al.,Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmidDNA (pDNA). A polynucleotide can comprise a conventional phosphodiesterbond or a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acidsequence” refer to any one or more nucleic acid segments, e.g., DNA orRNA fragments, present in a polynucleotide.

By an “isolated” nucleic acid or polynucleotide is intended any form ofthe nucleic acid or polynucleotide that is separated from its nativeenvironment. For example, gel-purified polynucleotide, or a recombinantpolynucleotide encoding a polypeptide contained in a vector would beconsidered to be “isolated.” Also, a polynucleotide segment, e.g., a PCRproduct, that has been engineered to have restriction sites for cloningis considered to be “isolated.” Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in a non-native solution such as a buffer or saline.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofpolynucleotides, where the transcript is not one that would be found innature. Isolated polynucleotides or nucleic acids further include suchmolecules produced synthetically. In addition, polynucleotide or anucleic acid can be or can include a regulatory element such as apromoter, ribosome binding site, or a transcription terminator.

As used herein, a “non-naturally occurring” polynucleotide, or anygrammatical variants thereof, is a conditional definition thatexplicitly excludes, but only excludes, those forms of thepolynucleotide that are well-understood by persons of ordinary skill inthe art as being “naturally-occurring,” or that are, or that might be atany time, determined or interpreted by a judge or an administrative orjudicial body to be, “naturally-occurring.”

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g., on a single vector, or in separate polynucleotideconstructs, e.g., on separate (different) vectors. Furthermore, anyvector can contain a single coding region, or can comprise two or morecoding regions, e.g., a single vector can separately encode animmunoglobulin heavy chain variable region and an immunoglobulin lightchain variable region. In addition, a vector, polynucleotide, or nucleicacid can include heterologous coding regions, either fused or unfused toanother coding region. Heterologous coding regions include withoutlimitation, those encoding specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally can include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter can be a cell-specific promoter thatdirects substantial transcription of the DNA in predetermined cells.Other transcription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit ß-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in theform of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

Polynucleotide and nucleic acid coding regions can be associated withadditional coding regions that encode secretory or signal peptides,which direct the secretion of a polypeptide encoded by a polynucleotideas disclosed herein. According to the signal hypothesis, proteinssecreted by mammalian cells have a signal peptide or secretory leadersequence that is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells can have a signal peptidefused to the N-terminus of the polypeptide, which is cleaved from thecomplete or “full length” polypeptide to produce a secreted or “mature”form of the polypeptide. In certain embodiments, the native signalpeptide, e.g., an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, can be used. Forexample, the wild-type leader sequence can be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseß-glucuronidase.

Disclosed herein are certain binding molecules, or antigen bindingfragments, variants, or derivatives thereof. Unless specificallyreferring to full-sized antibodies, the term “binding molecule”encompasses full-sized antibodies as well as antigen binding subunits,fragments, variants, analogs, or derivatives of such antibodies, e.g.,engineered antibody molecules or fragments that bind antigen in a mannersimilar to antibody molecules, but which use a different scaffold.

As used herein, the term “binding molecule” refers in its broadest senseto a molecule that specifically binds to a receptor, e.g., an epitope oran antigenic determinant. As described further herein, a bindingmolecule can comprise one or more “antigen binding domains” describedherein. A non-limiting example of a binding molecule is an antibody orfragment thereof that retains antigen-specific binding.

The terms “binding domain” and “antigen binding domain” are usedinterchangeably herein and refer to a region of a binding molecule thatis necessary and sufficient to specifically bind to an epitope. Forexample, an “Fv,” e.g., a variable heavy chain and variable light chainof an antibody, either as two separate polypeptide subunits or as asingle chain, is considered to be a “binding domain.”

Other antigen binding domains include, without limitation, the variableheavy chain (VHH) of an antibody derived from a camelid species, or siximmunoglobulin complementarity determining regions (CDRs) expressed in afibronectin scaffold. A “binding molecule” as described herein caninclude one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more “antigen binding domains.”

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein) includes at least the variable region of a heavy chain(for camelid species) or at least the variable regions of a heavy chainand a light chain. Basic immunoglobulin structures in vertebrate systemsare relatively well understood. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).Unless otherwise stated, the term “antibody” encompasses anythingranging from a small antigen binding fragment of an antibody to a fullsized antibody, e.g., an IgG antibody that includes two complete heavychains and two complete light chains, an IgA antibody that includes fourcomplete heavy chains and four complete light chains and can include aJ-chain and/or a secretory component, or an IgM antibody that includesten or twelve complete heavy chains and ten or twelve complete lightchains and can include a J-chain.

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4 or α1-α2)).It is the nature of this chain that determines the “class” of theantibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulinsubclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂, etc. arewell characterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of this disclosure.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. The basicstructure of certain antibodies, e.g., IgG antibodies, includes twoheavy chain subunits and two light chain subunits covalently connectedvia disulfide bonds to form a “Y” structure, also referred to herein asan “H2L2” structure, or a “binding unit.”

The term “binding unit” is used herein to refer to the portion of abinding molecule, e.g., an antibody or antigen binding fragment thereofthat corresponds to a standard immunoglobulin structure, i.e., two heavychains or fragments thereof and two light chains or fragments thereof,or two heavy chains or fragments thereof derived, e.g., from a camelidor condricthoid antibody. In certain aspects, e.g., where the bindingmolecule is a bivalent, single binding unit IgG antibody or antigenbinding fragment thereof, the terms “binding molecule” and “bindingunit” are equivalent. In other aspects, e.g., where the binding moleculeis an IgA dimer, an IgM pentamer, or an IgM hexamer, the bindingmolecule is “multimeric” and comprises two or more “binding units.” Twoin the case of an IgA dimer, or five or six in the case of an IgMpentamer or hexamer, respectively. A binding unit need not includefull-length antibody heavy and light chains, but will typically bebivalent, i.e., will include two “antigen binding domains,” as definedbelow. Certain binding molecules provided in this disclosure arepentameric or hexameric, and include five or six bivalent binding unitsthat include IgM constant regions or fragments thereof.

As used herein, a binding molecule comprising two or more binding units,e.g., two, five, or six binding units, can be referred to as“multimeric.” The term “multimeric” means possessing more than one unit.Thus, for example, a “multimeric binding molecule” will possess morethan one binding unit. A multimeric binding molecule can possess two,three four, five or even six or more binding units. A “dimeric bindingmolecule” includes two binding units and is typically a dimeric IgAmolecule that further comprises a J-chain. A “pentameric bindingmolecule” is typically a pentameric IgM binding molecule that furthercomprises a J-chain. A “hexameric binding molecule” is typically ahexameric IgM binding molecule. In contrast, a “single binding unitmolecule” can be, e.g., an IgG antibody.

The terms “wild-type (WT) J-chain,” “native sequence J-chain” or “nativeJ-chain” as used herein refer to a J-chain of native sequence IgM or IgAantibodies of any animal species, including mature human J-chain, theamino acid sequence of which is presented as SEQ ID NO: 2.

The term “modified J-chain” is used herein to refer to variants of anative J-chain polypeptide. A modified J-chain can be a full-lengthmature J-chain polypeptide or a functional fragment thereof, and caninclude, without limitation, amino acid insertions, deletions,substitutions, non-amino acid modifications such as glycosylation orlipidation. Moreover, a modified J-chain can include a heterologousmoiety such as a binding moiety, either introduced into the J-chainamino acid sequence as a fusion protein, or attached or conjugated byother techniques, such as disulfide bonding, or chemical conjugation. Amodified J-chain is typically functional, in that the modifications donot interfere with efficient polymerization of IgM or IgA and binding ofsuch polymers to a target. Exemplary modified J-chains are describedelsewhere herein and in PCT Publication No. WO 2015/153912, which isincorporated herein by reference in its entirety. The term “modifiedhuman J-chain” encompasses, without limitation, a native sequence humanJ-chain of the amino acid sequence of SEQ ID NO: 2 or functionalfragment thereof modified as above, e.g., by the introduction of aheterologous moiety, e.g., a heterologous polypeptide, e.g., anadditional desired binding domain.

The terms “valency,” “bivalent,” “multivalent” and grammaticalequivalents, refer to the number of antigen binding domains in givenbinding molecule or binding unit. For example, the terms “bivalent”,“tetravalent”, and “hexavalent” in reference to a given bindingmolecule, e.g., an IgM antibody or fragment thereof, denote the presenceof two antigen binding domains, four antigen binding domains, and sixantigen binding domains, respectively. In a typical IgM-derived bindingmolecule, each binding unit is bivalent, whereas the binding moleculeitself can have 10 or 12 valencies. A bivalent or multivalent bindingmolecule can be monospecific, i.e., all of the antigen binding domainsare the same, or can be bispecific or multispecific, e.g., where two ormore antigen binding domains are different, e.g., bind to differentepitopes on the same antigen, or bind to entirely different antigens.

The term “epitope” includes any molecular determinant capable ofspecific binding to an antibody. In certain aspects, an epitope caninclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl, or sulfonyl, and, in certainaspects, can have three dimensional structural characteristics, and orspecific charge characteristics. An epitope is a region of a target thatis bound by an antibody.

“Multispecific binding molecules or antibodies” or “bispecific bindingmolecules or antibodies” refer to binding molecules, antibodies, orantigen binding fragments thereof that have the ability to specificallybind to two or more different epitopes on the same or differenttarget(s). “Monospecific” refers to the ability to bind only oneepitope.

The term “target” is used in the broadest sense to include substancesthat can be bound by a binding molecule. A target can be, e.g., apolypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule.Moreover, a “target” can, for example, be a cell, an organ, or anorganism that comprises an epitope bound that can be bound by a bindingmolecule.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variableregions (which can be called “variable domains” interchangeably herein)of both the variable light (VL) and variable heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (e.g., CH1, CH2 orCH3) confer biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 (or CH4 in the case ofIgM) and CL domains are at the carboxy-terminus of the heavy and lightchain, respectively.

A “full length IgM antibody heavy chain” is a polypeptide that includes,in N-terminal to C-terminal direction, an antibody heavy chain variableregion (VH), an antibody constant heavy chain constant domain 1 (CM1 orCμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), anantibody heavy chain constant domain 3 (CM3 or Cμ3), and an antibodyheavy chain constant domain 4 (CM4 or Cμ4), which can also include atailpiece.

A “full length IgA antibody heavy chain” is a polypeptide that includes,in N-terminal to C-terminal direction, an antibody heavy chain variableregion (V_(H)), an antibody constant heavy chain constant domain 1 (CA1or Cα1), an antibody heavy chain constant domain 2 (CA2 or Cα2), anantibody heavy chain constant domain 3 (CA3 or Cα3), and a tailpiece andcan be either an IgA1 or IgA2. The structure of monomeric, dimeric(J-chain-containing) and secretory IgA is described, e.g., in Woof, J Mand Russell, M W, Mucosal Immunology 4:590-597 (2011).

Both IgA and IgM possess an 18-amino acid extension in the C terminuscalled the “tail-piece” (tp). The IgM (μtp) and IgA (αtp) tail-piecesdiffer at seven amino acid positions. The IgM and IgA tail-piece ishighly conserved among various animal species. The conserved penultimatecysteine residue in the IgA and IgM tail-pieces has been demonstrated tobe involved in polymerization. Both tail-pieces contain an N-linkedcarbohydrate addition site, the presence of which is required for dimerformation in IgA and J-chain incorporation and pentamer formation inIgM. However, the structure and composition of the N-linkedcarbohydrates in the tail-pieces differ, suggesting differences in theaccessibility of the glycans to processing by glycosyltransferases.

As indicated above, a variable region, i.e., the “antigen bindingdomain,” allows the binding molecule to selectively recognize andspecifically bind epitopes on antigens. That is, the VL domain and VHdomain, or subset of the complementarity determining regions (CDRs), ofa binding molecule, e.g., an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. Certain antibodies form larger structures. Forexample, IgA can form a molecule that includes two H2L2 units and aJ-chain, all covalently connected via disulfide bonds, and IgM can forma pentameric or hexameric molecule that includes five or six H2L2 unitsand, in some embodiments, a J-chain covalently connected via disulfidebonds. In certain embodiments, polymeric IgA and IgM molecules can alsocontain a secretory component that can also be covalently connected viadisulfide bonds. Further, it is known that both IgA and pentameric IgMbind to the polymeric immunoglobulin receptor (pIgR) and are secretedafter binding. (See, Mostov K. E., Ann. Rev. Immunol., 12:63-84, 1994,page 65).

The six “complementarity determining regions” or “CDRs” present in anantibody antigen binding domain are short, non-contiguous sequences ofamino acids that are specifically positioned to form the antigen bindingdomain as the antibody assumes its three dimensional configuration in anaqueous environment. The remainder of the amino acids in the antigenbinding domain, referred to as “framework” regions, show lessinter-molecular variability. The framework regions largely adopt aβ-sheet conformation and the CDRs form loops that connect, and in somecases form part of, the β-sheet structure. Thus, framework regions actto form a scaffold that provides for positioning the CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto its cognate epitope. The amino acids that make up the CDRs and theframework regions, respectively, can be readily identified for any givenheavy or light chain variable region by one of ordinary skill in theart, since they have been defined in various different ways (see,“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1983); and Chothia andLesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated hereinby reference in their entireties).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. These particular regionshave been described, for example, by Kabat et al., U.S. Dept. of Healthand Human Services, “Sequences of Proteins of Immunological Interest”(1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), whichare incorporated herein by reference. The Kabat and Chothia definitionsinclude overlapping or subsets of amino acids when compared against eachother. Nevertheless, application of either definition (or otherdefinitions known to those of ordinary skill in the art) to refer to aCDR of an antibody or variant thereof is intended to be within the scopeof the term as defined and used herein, unless otherwise indicated. Theappropriate amino acids that encompass the CDRs as defined by each ofthe above cited references are set forth below in Table 1 as acomparison. The exact amino acid numbers that encompass a particular CDRwill vary depending on the sequence and size of the CDR. Those skilledin the art can routinely determine that amino acids comprise aparticular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions* Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 *Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Rabat et al. (seebelow).

Immunoglobulin variable domains can also be analyzed, e.g., using theIMGT information system (www://imgt.cines.fr/) (IMGT®/V-Quest) toidentify variable region segments, including CDRs. (See, e.g., Brochetet al., Nucl. Acids Res., 36:W503-508, 2008).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless use of the Kabat numbering system is explicitly noted, however,consecutive numbering is used for all amino acid sequences in thisdisclosure.

Binding molecules, e.g., antibodies or antigen binding fragments,variants, or derivatives thereof include, but are not limited to,polyclonal, monoclonal, human, humanized, or chimeric antibodies, singlechain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), single domain antibodies such as camelidVHH antibodies, fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library. ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulinor antibody molecules encompassed by this disclosure can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Alsocontemplated are immunoglobulin new antigen receptor (IgNAR) isotypesthat are bivalent and comprise a single chain that includes an IgNARvariable domain (VNAR). (See, Walsh et al., Virology 411:132-141, 2011).

By “specifically binds,” it is generally meant that a binding molecule,e.g., an antibody or fragment, variant, or derivative thereof binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, a binding molecule is said to“specifically bind” to an epitope when it binds to that epitope, via itsantigen binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain binding molecule binds to a certainepitope. For example, binding molecule “A” can be deemed to have ahigher specificity for a given epitope than binding molecule “B,” orbinding molecule “A” can be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof disclosed herein can be said to bind a target antigenwith an off rate (k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻²sec⁻¹, 5×10⁻³ sec⁻¹, 10⁻³ sec⁻¹, 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹,or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen binding fragment,variant, or derivative disclosed herein can be said to bind a targetantigen with an on rate (k(on)) of greater than or equal to 10³ M⁻¹sec⁻¹, 5×10³M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹, 5×10⁴M⁻¹ sec⁻¹, 10⁵ M⁻¹ sec⁻¹,5×10⁵ M⁻¹ sec⁻¹, 10⁶M⁻¹ sec⁻¹, or 5×10⁶M⁻¹ sec⁻¹ or 10⁷M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof is said to competitively inhibit binding of areference antibody or antigen binding fragment to a given epitope if itpreferentially binds to that epitope to the extent that it blocks, tosome degree, binding of the reference antibody or antigen bindingfragment to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Abinding molecule can be said to competitively inhibit binding of thereference antibody or antigen binding fragment to a given epitope by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with one or more antigen bindingdomains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population of antigenbinding domains and an antigen. See, e.g., Harlow at pages 29-34.Avidity is related to both the affinity of individual antigen bindingdomains in the population with specific epitopes, and also the valenciesof the immunoglobulins and the antigen. For example, the interactionbetween a bivalent monoclonal antibody and an antigen with a highlyrepeating epitope structure, such as a polymer, would be one of highavidity. An interaction between a between a bivalent monoclonal antibodywith a receptor present at a high density on a cell surface would alsobe of high avidity.

Binding molecules or antigen binding fragments, variants or derivativesthereof as disclosed herein can also be described or specified in termsof their cross-reactivity. As used herein, the term “cross-reactivity”refers to the ability of a binding molecule, e.g., an antibody orfragment, variant, or derivative thereof, specific for one antigen, toreact with a second antigen; a measure of relatedness between twodifferent antigenic substances. Thus, a binding molecule is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof can also be described or specified in terms of itsbinding affinity to an antigen. For example, a binding molecule can bindto an antigen with a dissociation constant or K_(D) no greater than5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵M, 10⁻⁵M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³M,10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

Antibody fragments including single-chain antibodies or other antigenbinding domains can exist alone or in combination with one or more ofthe following: hinge region, CH1, CH2, CH3, or CH4 domains, J-chain, orsecretory component. Also included are antigen binding fragments thatcan include any combination of variable region(s) with one or more of ahinge region, CH1, CH2, CH3, or CH4 domains, a J-chain, or a secretorycomponent. Binding molecules, e.g., antibodies, or antigen bindingfragments thereof can be from any animal origin including birds andmammals. The antibodies can be human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region can be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and can in someinstances express endogenous immunoglobulins and some not, as describedinfra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati etal.

As used herein, the term “heavy chain subunit” or “heavy chain domain”includes amino acid sequences derived from an immunoglobulin heavychain, a binding molecule, e.g., an antibody comprising a heavy chainsubunit can include at least one of: a VH domain, a CH1 domain, a hinge(e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, aCH3 domain, a CH4 domain, or a variant or fragment thereof. For example,a binding molecule, e.g., an antibody or fragment, variant, orderivative thereof can include, in addition to a VH domain, a CH1domain; CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, ahinge domain, a CH2 domain, and a CH3 domain. In certain aspects abinding molecule, e.g., an antibody or fragment, variant, or derivativethereof can include, in addition to a VH domain, a CH3 domain and a CH4domain; or a CH3 domain, a CH4 domain, and a J-chain. Further, a bindingmolecule for use in the disclosure can lack certain constant regionportions, e.g., all or part of a CH2 domain. It will be understood byone of ordinary skill in the art that these domains (e.g., the heavychain subunit) can be modified such that they vary in amino acidsequence from the original immunoglobulin molecule.

The heavy chain subunits of a binding molecule, e.g., an antibody orfragment thereof, can include domains derived from differentimmunoglobulin molecules. For example, a heavy chain subunit of apolypeptide can include a CH1 domain derived from an IgG1 molecule and ahinge region derived from an IgG3 molecule. In another example, a heavychain subunit can include a hinge region derived, in part, from an IgG1molecule and, in part, from an IgG3 molecule. In another example, aheavy chain subunit can comprise a chimeric hinge derived, in part, froman IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain subunit” or “light chain domain”includes amino acid sequences derived from an immunoglobulin lightchain. The light chain subunit includes at least one of a VL or CL(e.g., Cκ or Cλ) domain.

Binding molecules, e.g., antibodies or antigen binding fragments,variants, or derivatives thereof can be described or specified in termsof the epitope(s) or portion(s) of an antigen that they recognize orspecifically bind. The portion of a target antigen that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target antigen can comprisea single epitope or at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH region” or “VHdomain” includes the amino terminal variable domain of an immunoglobulinheavy chain and the term “CH1 domain” includes the first (most aminoterminal) constant region domain of an immunoglobulin heavy chain,extending, e.g., from about amino acid 114 to about amino acid 223 of anIgG antibody using conventional numbering schemes (amino acids 114 to223, Kabat numbering system; and amino acids 118-215, EU numberingsystem; see Kabat E A et al., op. .cit). The CH1 domain is adjacent tothe VH domain and is amino terminal to the hinge region of a typical IgGheavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about amino acid 244 to aminoacid 360 of an IgG antibody using conventional numbering schemes (aminoacids 244 to 360, Kabat numbering system; and amino acids 231-340, EUnumbering system; see Kabat E A et al., op. .cit). The CH3 domainextends from the CH2 domain to the C-terminal of the IgG molecule andcomprises approximately 108 amino acids. Certain immunoglobulin classes,e.g., IgM, further include a CH4 region.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain in IgG, IgA,and IgD heavy chains. This hinge region comprises approximately 25 aminoacids and is flexible, thus allowing the two N-terminal antigen bindingregions to move independently.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In certain IgG molecules, the CH1 and CL regions are linked by adisulfide bond and the two heavy chains are linked by two disulfidebonds at positions corresponding to 239 and 242 using the Kabatnumbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” refers to an antibody inwhich the immunoreactive region or site is obtained or derived from afirst species and the constant region (which can be intact, partial ormodified) is obtained from a second species. In some embodiments thetarget binding region or site will be from a non-human source (e.g.mouse or primate) and the constant region is human.

The terms “multispecific antibody, or “bispecific antibody” refer to anantibody that has antigen binding domains that are specific for two ormore different epitopes within a single antibody molecule (or “bindingunit”). Other binding molecules in addition to the canonical antibodystructure can be constructed with two different binding specificities.Epitope binding by bispecific or multispecific antibodies can besimultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies.Bispecific antibodies can also be constructed by recombinant means.(Ströhlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely,IDrugs. 13:543-9 (2010)). A bispecific antibody can also be a diabody.Thus, a bispecific binding molecule that is multimeric could potentiallypossess several different antigen binding domains, each with a differentspecificity. For instance, an IgM binding molecule would be consideredmultimeric, containing five or six binding units, and each binding unitpossessing possibly two antigen binding domains. Such an IgM bindingmolecule could therefore have as many as two, three, four, five, six,seven, eight, nine, ten, eleven, or even twelve different specificities,since each antigen binding domain can bind a different, distinguishableepitope. In certain aspects each binding unit is a monospecific H2L2structure. Bispecific and multi-specific IgM and IgA binding molecules,including antibodies, are described, for example, in PCT Publication No.WO 2015/053887 and PCT Publication No. WO 2015/120474, the entirecontents of which are hereby expressly incorporated by reference. Incertain aspects a heterologous binding domain can be associated with aJ-chain, as described in PCT Publication No. WO 2015/153912, andelsewhere herein.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more amino acids ineither the CDR or framework regions. In certain aspects entire CDRs froman antibody of known specificity can be grafted into the frameworkregions of a heterologous antibody. Although alternate CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, CDRs can also bederived from an antibody of different class, e.g., from an antibody froma different species. An engineered antibody in which one or more “donor”CDRs from a non-human antibody of known specificity are grafted into ahuman heavy or light chain framework region is referred to herein as a“humanized antibody.” In certain aspects not all of the CDRs arereplaced with the complete CDRs from the donor variable region and yetthe antigen binding capacity of the donor can still be transferred tothe recipient variable domains. Given the explanations set forth in,e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, itwill be well within the competence of those skilled in the art, eitherby carrying out routine experimentation or by trial and error testing toobtain a functional engineered or humanized antibody.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” or othergrammatical equivalents can be used interchangeably. These terms referto the joining together of two more elements or components, by whatevermeans including chemical conjugation or recombinant means. An “in-framefusion” refers to the joining of two or more polynucleotide open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the translational reading frame of the original ORFs. Thus, arecombinant fusion protein is a single protein containing two or moresegments that correspond to polypeptides encoded by the original ORFs(which segments are not normally so joined in nature.) Although thereading frame is thus made continuous throughout the fused segments, thesegments can be physically or spatially separated by, for example,in-frame linker sequence. For example, polynucleotides encoding the CDRsof an immunoglobulin variable region can be fused, in-frame, but beseparated by a polynucleotide encoding at least one immunoglobulinframework region or additional CDR regions, as long as the “fused” CDRsare co-translated as part of a continuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which amino acids that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

A portion of a polypeptide that is “amino-terminal” or “N-terminal” toanother portion of a polypeptide is that portion that comes earlier inthe sequential polypeptide chain. Similarly a portion of a polypeptidethat is “carboxy-terminal” or “C-terminal” to another portion of apolypeptide is that portion that comes later in the sequentialpolypeptide chain. For example in a typical antibody, the variabledomain is “N-terminal” to the constant region, and the constant regionis “C-terminal” to the variable domain.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide that istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt or slow the progression of an existingdiagnosed pathologic condition or disorder. Terms such as “prevent,”“prevention,” “avoid,” “deterrence” and the like refer to prophylacticor preventative measures that prevent the development of an undiagnosedtargeted pathologic condition or disorder. Thus, “those in need oftreatment” can include those already with the disorder; those prone tohave the disorder; and those in whom the disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromtherapy” and “an animal in need of treatment” includes subjects, such asmammalian subjects, that would benefit from administration of a bindingmolecule such as an antibody, comprising one or more antigen bindingdomains. Such binding molecules, e.g., antibodies, can be used, e.g.,for a diagnostic procedures and/or for treatment or prevention of adisease.

Multimeric Binding Molecules

This disclosure provides a multimeric HIV binding molecule, i.e., abinding molecule possessing at least two, e.g., two, five, or six“binding units” as defined herein, where at least one antigen bindingdomain of the multimeric binding molecule can specifically bind to anHIV antigen, e.g., an HIV protein, e.g., gp120 and/or gp41. Exemplaryepitopes on gp120 and/or gp41 that can be bound by a multimeric HIVbinding molecule provided herein, include, without limitation, theimmunodominant region of gp41, the membrane proximal external region(MPER), the CD4 binding site, the variable region 1/variable Region 2(V1/V2) loop, the glycan-variable region 3 (V3) loop, and/or anycarbohydrates associated with these regions. Exemplary multimericbinding molecules include, but are not limited to, an IgM bindingmolecule with five or six binding units (a pentameric or hexamericbinding molecule), or an IgA binding molecule with two binding units.

A multimeric binding molecule as provided herein can have improvedbinding characteristics or biological activity as compared to a bindingmolecule composed of a single binding unit, e.g., a bivalent IgGantibody. In some embodiments, a multimeric binding molecule as providedherein can more potently neutralize HIV, bind and neutralize morediverse HIV variants or clades, enhance viral clearance, improve tissuedistribution (e.g., to mucosal surfaces), and/or be more potent inpreventing, controlling or treating HIV infection than a correspondingreference single binding unit molecule comprising only two HIV antigenbinding domains. In certain embodiments a multimeric binding molecule asprovided herein can be more potent in controlling HIV infectivity andgrowth as compared with a corresponding reference single binding unitmolecule comprising only two HIV antigen binding domains. In certainembodiments a multimeric binding molecule as provided herein can be usedto treat chronic infection, e.g., by binding to and/or effectingantibody and/or cell-mediated killing of HIV infected cells, e.g.,reservoir cells that express extremely low levels of HIV antigens ontheir surface. In certain embodiments, the multimeric binding moleculecan be more effective at activating and killing such HIV-infected cellsor killing such cells after activation with an independent activatingagent such as an effector cell. In certain embodiments, a multimericbinding molecule as provided herein can provide equivalent benefit at alower dosage than that of a corresponding reference single binding unitmolecule comprising only two HIV antigen binding domains. In certainembodiments administration of a multimeric binding molecule as providedherein can allow for reduced or modified dosages of other retroviraltherapies, e.g., ART. See, e.g., Example 7 below. The term“corresponding reference single binding unit molecule” refers to abinding molecule composed of a single binding unit, which has one or twoHIV antigen binding domains similar or identical to one or more HIVantigen binding domains of a dimeric, pentameric, or hexameric HIVbinding molecule, e.g., an IgM antibody provided herein.

In certain aspects, a “corresponding reference single binding unitmolecule” is an IgG antibody comprising two identical antigen bindingdomains, where those antigen binding domains are identical to thosecontained in at least one binding unit, or at least two, three, four,five, or six binding units, of a dimeric, pentameric, or hexameric HIVbinding molecule, e.g., an IgM antibody provided herein.

The term “improved binding characteristics” is a non-limiting term thatcan apply to any characteristic of the multimeric binding molecule thatis improved or distinctive relative to a monomeric binding molecule. Amultimeric binding molecule can, e.g., neutralize HIV in infected cells,e.g., cells in a human infected with HIV, and when administered to anindividual in need thereof, can exhibit an activity that is empiricallydetermined to be stronger, more potent, or require less binding moleculeby mass or molar equivalents, such as, but not limited to (i) reducingthe infectivity of an HIV virion, (ii) neutralizing more diverse HIVvariants or clades, (iii) reducing the number of HIV-infected cells(including reservoir cells), (iv) preventing HIV infection, (v)enhancing viral clearance, and/or (vi) improving the signs and symptomsof HIV infection, as compared with a corresponding reference singlebinding unit molecule (e.g., an IgG molecule) that possesses antigenbinding domains similar or identical in sequence to those of a dimeric,pentameric, or hexameric HIV binding molecule, e.g., an IgM antibodyprovided herein.

A corresponding reference single binding unit molecule as referred toabove can be an IgG binding molecule. The reference IgG binding moleculecan be of any isotype, such as IgG1, IgG2, IgG3, or IgG4, etc. Thereference binding molecule is typically from the same animal. Thus ifthe multimeric binding molecule is human, the corresponding referencesingle binding unit molecule would typically also be human. Conversely,if the multimeric binding molecule is a rabbit binding molecule, thecorresponding reference single binding unit molecule would also be arabbit binding molecule.

IgM Binding Molecules

IgM is the first immunoglobulin produced by B cells in response tostimulation by antigen, and is present at around 1.5 mg/ml in serum witha half-life of 5 days. IgM is typically multimeric, e.g., a pentamericor hexameric molecule. Thus, IgM molecules are “multimeric” bindingmolecules. Each of the five, or six, IgM binding units includes twolight and two heavy chains. While IgG contains three heavy chainconstant domains (CH1, CH2 and CH3), as explained above, the heavy (μ)chain of IgM additionally contains a fourth constant domain (CH4), thatincludes a C-terminal “tailpiece.” The human IgM constant regiontypically comprises the amino acid sequence SEQ ID NO: 1. The human Cμ1region ranges from about amino acid 5 to about amino acid 102 of SEQ IDNO: 1; the human Cμ2 region ranges from about amino acid 114 to aboutamino acid 205 of SEQ ID NO: 1, the human Cμ3 region ranges from aboutamino acid 224 to about amino acid 319 of SEQ ID NO: 1, the Cμ 4 regionranges from about amino acid 329 to about amino acid 430 of SEQ ID NO:1, and the tailpiece ranges from about amino acid 431 to about aminoacid 453 of SEQ ID NO: 1. The amino acid sequence of the human IgMconstant region (SEQ ID NO: 1) is provided below:

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGT CY

An IgM binding molecule can comprise five binding units (each an “IgMbinding unit”) that can form a complex with an additional smallpolypeptide chain (the J-chain) to form a pentameric IgM bindingmolecule. The human J-chain comprises the amino acid sequence SEQ ID NO:2. As described elsewhere herein the J-chain can be a variant J-chaincomprising, e.g., a binding moiety such as an ScFv or a camelidantibody. Without the J-chain, IgM binding units typically assemble intoa hexameric IgM binding molecule. While not wishing to be bound bytheory, the assembly of IgM binding units into a hexameric or pentamericbinding molecule is thought to involve the Cμ3 and Cμ4 domains.Accordingly, a hexameric or pentameric IgM binding molecule provided inthis disclosure typically includes IgM constant regions that include atleast the Cμ3 and Cμ4 domains. The amino acid sequence of the humanJ-chain (SEQ ID NO: 2) is provided below:

MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMV ETALTPDACYPD

The signal peptide (amino acids 1 to 22 of SEQ ID NO: 2) is doubleunderlined, the mature J-chain sequence is amino acids 23 to 159 of SEQID NO: 2.

An IgM heavy chain constant region can additionally include a Cμ2 domainor a fragment thereof, a Cμ1 domain or a fragment thereof, and/or otherIgM heavy chain domains. In certain aspects, a binding molecule asprovided herein can include a complete IgM heavy (μ) chain constantdomain, e.g., SEQ ID NO: 1, or a variant, derivative, or analog thereof.

Pentameric or Hexameric HIV Binding Molecules

This disclosure provides a pentameric or hexameric HIV binding molecule,a binding molecule that has five or six IgM-derived “binding units” asdefined herein, which can specifically bind to an HIV antigen, e.g., anHIV protein, e.g., the HIV spike protein. In certain aspects, eachbinding unit includes two IgM heavy chain constant regions or fragmentsthereof. In certain aspects, the two IgM heavy chain constant regionsare human heavy chain constant regions. In certain aspects, the antigenbinding domains in the IgM binding molecule are human in origin, orhumanized, or a combination thereof. A pentameric or hexameric IgMbinding molecule as provided herein can have improved bindingcharacteristics or biological activity as compared to a binding moleculecomposed of a single binding unit, e.g., a bivalent IgG-derivedantibody. In some embodiments, a pentameric or hexameric bindingmolecule provided herein can, e.g., through increased avidity oraffinity, or enhanced effector functions, be more potent in targetingchronic infections, e.g., by binding to and/or effectingcomplement-mediated killing of HIV reservoir cells, as compared with acorresponding reference single binding unit molecule containing only twoHIV-specific antigen binding domains.

A pentameric or hexameric HIV binding molecule as provided herein canlikewise possess distinctive characteristics as compared to univalent ormultivalent binding molecules composed of synthetic or chimericstructures. For example, use of human IgM constant regions can affordreduced immunogenicity and thus increased safety relative to a bindingmolecule containing chimeric constant regions or synthetic structures.Moreover, an IgM-based binding molecule can consistently form hexamericor pentameric oligomers resulting in a more homogeneous expressionproduct. Superior complement fixation can also be an advantageouseffector function of IgM-based binding molecules.

The reference single binding unit referred to above can be an IgGbinding unit. The reference IgG binding unit can be of any isotype, suchas IgG1, IgG2, IgG3, or IgG4, etc. The reference binding unit istypically from the same animal. Thus if the multimeric binding moleculeis human, the reference single binding unit would also be human, but notnecessarily human. That is, the reference single binding unit can be ahumanized antibody of the IgG type. Conversely, if the multimericbinding molecule is a rabbit binding molecule, the reference singlebinding unit would also be a rabbit binding unit. Further, if themultimeric binding molecule is comprised of one or more binding unitfragments, then the reference single binding unit would also be anequivalent single binding unit fragment. In other words, the referencesingle binding unit is otherwise identical in sequence and structure tothe binding units contained in the multimeric binding molecule exceptthat the reference single binding unit is an equivalent single bindingunit.

In certain aspects, the disclosure provides a pentameric or hexamericbinding molecule comprising five or six binding units, respectively,where each binding unit includes two IgM heavy chain constant regions orfragments thereof. In certain aspects, the two IgM heavy chain constantregions are human heavy chain constant regions. In some embodiments, theantigen binding domains in the IgM binding molecule are human in origin,or humanized, or a combination thereof.

Where the multimeric binding molecule provided herein is pentameric, thebinding molecule can further comprise a J-chain, or functional fragmentthereof, or variant thereof. Where the pentameric IgM binding moleculecontains a J-chain, the J-chain can be of the same species as the IgMbinding molecule. That is, if the pentameric IgM binding molecule ishuman, the J-chain can also be human. In certain aspects, the J-chaincan be a modified J-chain comprising a heterologous moiety or one ormore heterologous moieties, e.g., a heterologous polypeptide sequence,e.g., an additional desired binding domain introduced into the nativesequence. In certain aspects the additional binding domain specificallybinds to CD3, e.g., CD3ε, or CD16.

In certain aspects each of the two IgM heavy chain constant regions in abinding unit is associated with an antigen binding domain, for examplean Fv portion of an antibody, e.g., a VH and a VL of a human or murineantibody. In certain aspects, at least one antigen binding domain of abinding molecule as provided herein is a cross-reactive HIV antigenbinding domain, e.g., an antigen binding domain that can bind to an HIVantigen from two or more HIV types, Groups, or clades. In certainaspects, the antigen binding domain can bind to the HIV antigen fromboth HIV types (types 1 and 2). In certain aspects, the binding moleculecan bind to the HIV antigen from two or more Groups (M, N and O) ofHIV-2. In certain aspects, the antigen binding domain can specificallybind to the HIV antigen from two, three, four, or more HIV groups orclades. In certain aspects, the binding molecule can bind to the HIVspike protein, e.g., gp120 and/or gp41, of two or more HIV types,groups, or clades. Exemplary epitopes on gp120 and/or gp41 epitopesinclude, without limitation, gp41, e.g., the immunodominant region ofgp41, the MPER, the CD4 binding site, the V1/V2 loop, the V3 loop,and/or any carbohydrates associated with these regions

In other embodiments, each antigen binding domain of a pentameric orhexameric HIV binding molecule as provided herein can independently binda different antigen or different epitope on the same antigen. Thus, apentameric IgM binding molecule can bind as many as two, three, four,five, six, seven, eight, nine or even ten different antigens orepitopes, across different HIV groups, subtypes or clades. Likewise, ahexameric IgM binding molecule can bind as many as two, three, four,five, six, seven, eight, nine, ten, eleven, or twelve different antigensor epitopes, across different HIV groups, subtypes or clades.

In certain aspects, a pentameric or hexameric HIV binding molecule asprovided herein comprises at least one antigen binding domain that bindsto an epitope on the HIV spike protein, e.g., gp120 and/or gp41, of twoor more HIV types, groups, or clades. In other aspects, two or moreantigen binding domains of a pentameric or hexameric HIV bindingmolecule provided herein, e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, or twelve antigen binding domains, can bind totwo or more distinct HIV spike protein epitopes, e.g., theimmunodominant region of gp41, the MPER, the CD4 binding site, the V1/V2loop, the V3 loop, and/or any carbohydrates associated with theseregions. In some aspects, a pentameric or hexameric HIV binding moleculeas provided herein can comprise at least one antigen binding domain thatspecifically binds the HIV spike protein, and can comprise other antigenbinding domains that specifically bind to other HIV proteins, e.g., gag,pol, tat, rev, nef, vpr, vif, and/or vpu. Alternatively, all of theantigen binding domains can possess the same specificity, e.g., for aspecific epitope on the HIV spike protein. In one aspect, all bindingunits of the pentameric or hexameric HIV binding molecule specificallybind to the HIV spike protein, for example, a pentameric or hexamericHIV binding molecule can comprise ten or twelve antigen binding domainsthat bind to the same spike protein epitope. In certain aspects the tenor twelve antigen binding domains can be identical.

In certain aspects, a pentameric or hexameric HIV binding molecule asprovided herein can bind to an HIV virion particle, and/or can bind tothe surface of an HIV-infected cell. In certain aspects, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, at least eleven, orat least twelve antigen binding domains of the pentameric or hexamericHIV binding molecule can specifically bind to an HIV virion particle,and/or can bind to the surface of an HIV-infected cell.

An HIV antigen or epitope bound by a pentameric or hexameric HIV bindingmolecule provided herein can be any one or more of the HIV proteins,including the HIV spike protein, e.g., gp120 and/or gp41, e.g., theimmunodominant region of gp41, the MPER, the CD4 binding site, the V1/V2loop, the V3 loop, and/or any carbohydrates associated with theseregions. Moreover, a pentameric or hexameric HIV binding molecule asprovided herein can be multispecific, including antigen binding domainsthat specifically bind to two or more antigens, e.g., one or more HIVantigens or epitopes and one or more heterologous antigens or epitopes,or two or more HIV antigens or epitopes. For example in certain aspectsa multispecific hexameric or pentameric HIV binding molecule providedherein can include five or six binding units each comprising two antigenbinding domains, where at least two individual binding domains bind todifferent antigens or epitopes. In certain non-limiting aspects one ormore binding domains can bind to, e.g., an epitope within the CD4binding site of the spike protein, while one or more of the remainingbinding domains can bind to, e.g., the immunodominant region of gp41,another epitope within the CD4 binding site, an epitope within the MPERregion, the V1/V2 loop, the V3 loop, and/or any carbohydrates associatedwith these regions, or any other region of the spike protein (exemplarybinding domains are provided in Table 3), or an epitope of another HIVprotein. In another aspect, a multispecific IgM binding molecule cancomprise binding domains that are specific for different subsets of orindividual HIV groups or clades, thereby providing a binding moleculewith activity over a broader range of HIV viruses. Methods of makingbispecific and multi-specific IgM and IgA binding molecules, includingantibodies, are described, for example, in PCT Publication No. WO2015/053887 and PCT Publication No. WO 2015/120474, the entire contentsof which are hereby expressly incorporated by reference. In certainaspects a heterologous binding domain can be associated with a J-chain,as described in PCT Publication No. WO 2015/153912, and elsewhereherein.

IgA Binding Molecules

IgA plays a critical role in mucosal immunity, and comprises about 15%of total immunoglobulin produced. IgA is a monomeric or dimericmolecule. Dimeric IgA molecules are relatively smaller in size than IgMmolecules, but can also possess improved binding characteristicsrelative to a corresponding reference single binding unit molecule.Moreover, a dimeric IgA binding molecule can reach mucosal sitesproviding greater tissue distribution for the binding molecules providedherein. Likewise, a dimeric IgA-derived binding molecule as providedherein can possess binding characteristics or biological activity thatcan be distinguished from a binding molecule comprising five or sixbinding units, e.g., a hexameric or pentameric IgM-derived bindingmolecule as described elsewhere herein. For example, a dimeric bindingmolecule would be smaller, and could, for example, achieve better tissuepenetration. Dimeric IgA binding molecules can be manufactured byexpression in vitro to include two IgA monomers and a J-chain. Thedimeric J-chain-containing IgA molecules can then be administered to anindividual where the IgA molecules that migrate to mucous membranes ormucosal tissue can bind to, and form a complex with, a membrane-boundsecretory component (mSC, also referred to as the polymeric Ig receptor(pIgR)) produced by epithelial cells. The complex is translocated acrossepithelial cells and the mSC is cleaved, delivering sIgA to the mucosalsurfaces. (See, Kaetzel et al., Proc. Natl. Acad. Sci. USA88(19):8796-8800, 1991). Therefore, delivery of IgA to the blood streamcan provide targeting of mucosal tissues.

An IgA binding unit includes two light and two IgA heavy chains. IgAcontains three heavy chain constant domains (Cα1, Cα2 and Cα3), andincludes a C-terminal “tailpiece.” Human IgA has two subtypes, IgA1 andIgA2. The mature human IgA1 constant region typically comprises theamino acid sequence SEQ ID NO: 3, provided below:

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSV VMAEVDGTCYThe human Cα1 region ranges from about amino acid 6 to about amino acid98 of SEQ ID NO: 3; the human Cα2 region ranges from about amino acid125 to about amino acid 220 of SEQ ID NO: 3 the human Cα3 region rangesfrom about amino acid 228 to about amino acid 330 of SEQ ID NO: 3, andthe tailpiece ranges from about amino acid 331 to about amino acid 352of SEQ ID NO: 3.

The mature human IgA2 constant region typically comprises the amino acidsequence SEQ ID NO: 4, provided below:

ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCYThe human Cα1 region ranges from about amino acid 6 to about amino acid98 of SEQ ID NO: 4; the human Cα2 region ranges from about amino acid112 to about amino acid 207 of SEQ ID NO: 4, the human Cα3 region rangesfrom about amino acid 215 to about amino acid 317 of SEQ ID NO: 4, andthe tailpiece ranges from about amino acid 318 to about amino acid 340of SEQ ID NO: 4.

Two IgA binding units can form a complex with two additional polypeptidechains, the J-chain (SEQ ID NO: 2) and the secretory component (SEQ IDNO: 76) to form a secretory IgA (sIgA) antibody. The amino acid sequenceof the mature secretory component (SEQ ID NO: 76) is provided below:

KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPR

While not wishing to be bound by theory, the assembly of IgA bindingunits into a dimeric sIgA binding molecule is thought to involve the Cα3and tailpiece domains. Accordingly, a dimeric sIgA binding moleculeprovided in this disclosure typically includes IgA constant regions thatinclude at least the Cα3 and tailpiece domains. An IgA heavy chainconstant region can additionally include a Cα2 domain or a fragmentthereof, a Cα1 domain or a fragment thereof, and/or other IgA heavychain domains. In certain aspects, a binding molecule as provided hereincan include a complete IgA heavy (α) chain constant domain, e.g., SEQ IDNO: 3 or SEQ ID NO: 4, or a variant, derivative, or analog thereof.

Dimeric HIV Binding Molecules

This disclosure provides a dimeric binding molecule comprising two ormore IgA binding units as defined herein, where the dimeric HIV bindingmolecule comprises at least one antigen binding domain that canspecifically bind to an HIV antigen, e.g., an HIV protein, e.g., the HIVspike protein. As explained above in the context of a pentameric orhexameric HIV binding molecule, a dimeric HIV binding molecule asprovided herein can possess improved binding characteristics orbiological activity as compared to a binding molecule composed ofcorresponding reference single binding unit molecule, e.g., a bivalentIgG antibody. For example, a dimeric HIV binding molecule can effectimproved tissue penetration or tissue distribution, especially tomucosal surfaces. Thus, a dimeric IgA binding molecule processed fortransport and secretion could target HIV-reservoir cells in the GIT.

The reference single binding unit referred to above can be an IgGbinding unit. The reference IgG binding unit can be of any isotype, suchas IgG1, IgG2, IgG3, or IgG4, etc. The reference binding unit istypically from the same animal. Thus if the multimeric binding moleculeis human, the reference single binding unit would also be human, but notnecessarily human. That is, the reference single binding unit can be ahumanized antibody of the IgG type. Conversely, if the multimericbinding molecule is a rabbit binding molecule, the reference singlebinding unit would also be a rabbit binding unit. Further, if themultimeric binding molecule is comprised of one or more binding unitfragments, then the reference single binding unit would also be anequivalent single binding unit fragment. In other words, the referencesingle binding unit is otherwise identical in sequence and structure tothe binding units contained in the multimeric binding molecule exceptthat the reference single binding unit is an equivalent single bindingunit.

In certain aspects, the disclosure provides a dimeric HIV bindingmolecule comprising two binding units, where each binding unit includestwo IgA heavy chain constant regions or fragments thereof. In certainaspects, the two IgA heavy chain constant regions are human heavy chainconstant regions. The anti-HIV binding domain(s) can be, e.g., human orhumanized.

A dimeric HIV binding molecule as provided herein can further comprise aJ-chain, or fragment thereof, or variant thereof. A dimeric HIV bindingmolecule as provided herein can further comprise a secretory component,or fragment thereof, or variant thereof.

An IgA heavy chain constant region can include one or more of a Cα1domain, a Cα2 domain, and/or a Cα3 domain, provided that the constantregion can serve a desired function in the binding molecule, e.g.,associate with second IgA constant region to form an antigen bindingdomain, or associate with another IgA binding unit to form a dimericbinding molecule. In certain aspects the two IgA heavy chain constantregions or fragments thereof within an individual binding unit eachcomprise a Cα3 domain or fragment thereof, a tailpiece (TP) or fragmentthereof, or any combination of a Cα3 domain, a TP, or fragment thereof.In certain aspects the two IgA heavy chain constant regions or fragmentsthereof within an individual binding unit each further comprise a Cα2domain or fragment thereof, a Cα1 domain or fragment thereof, or a Cα1domain or fragment thereof and a Cα2 domain or fragment thereof.

In certain aspects each of the two IgA heavy chain constant regions in agiven binding unit is associated with an antigen binding domain, forexample an Fv portion of an antibody, e.g., a VH and a VL of a human ormurine antibody. In certain aspects, at least one antigen binding domainof a binding molecule as provided herein is a cross-reactive HIV antigenbinding domain, e.g., an antigen binding domain that can specificallybind to two, three, four, or more HIV types, subtypes or clades. Incertain aspects, the antigen binding domain can specifically bind to theHIV antigen from two, three, four, or more HIV groups or clades. Incertain aspects, the binding molecule can bind to the HIV spike protein,e.g., gp120 and/or gp41, of two or more HIV types, groups, or clades.Exemplary epitopes on gp120 and/or gp41 epitopes include, withoutlimitation, the immunodominant region of gp41, the MPER, the CD4 bindingsite, the V1/V2 loop, the V3 loop, and/or any carbohydrates associatedwith these regions.

In other embodiments, a dimeric HIV binding molecule of the presentdisclosure can comprise binding units wherein each binding unit canpossess two antigen binding domains, each with a different anddistinguishable specificity. Thus, a dimeric HIV binding molecule couldpossess as many as four different specificities.

In certain aspects, the antigen binding domain can bind to the HIVantigen from both HIV types (types 1 and 2). In certain aspects, thedimeric binding molecule can bind to the HIV antigen from two or moreGroups (M, N and O) of HIV-2. In certain aspects, the antigen bindingdomain can specifically bind to the HIV antigen from two, three, four,or more HIV groups or clades.

In other embodiments, each antigen binding domain of a dimeric HIVbinding molecule as provided herein can independently bind a differentantigen or different epitope on the same antigen. Thus, a dimeric HIVbinding molecule can bind as many as two, three, or four differentantigens or epitopes, across different HIV groups, subtypes or clades.

In certain aspects, a dimeric HIV binding molecule as provided hereincomprises at least one antigen binding domain that binds to an epitopeon the HIV spike protein, e.g., gp120 and/or gp41, of two or more HIVtypes, groups, or clades. In other aspects, two or more antigen bindingdomains of a dimeric HIV binding molecule provided herein, e.g., two,three, or four antigen binding domains, can bind to two or more distinctHIV spike protein epitopes, e.g., the immunodominant region of gp41, theMPER, the CD4 binding site, the V1/V2 loop, the V3 loop, and/or anycarbohydrates associated with these regions. In some aspects, a dimericHIV binding molecule as provided herein can comprise at least oneantigen binding domain that specifically binds to the HIV spike protein,and can comprise other antigen binding domains that specifically bindother HIV proteins, e.g., gag, pol, tat, rev, nef, vpr, vif, and/or vpu.Alternatively, all of the antigen binding domains can possess the samespecificity, e.g., for a specific epitope on the HIV spike protein. Inone aspect, all binding units of the dimeric HIV binding moleculespecifically bind to the HIV spike protein; for example, a dimeric HIVbinding molecule can comprise four antigen binding domains that bind tothe same spike protein epitope. In certain aspects the four antigenbinding domains can be identical.

In certain aspects, a dimeric HIV binding molecule as provided hereincan bind to an HIV virion particle, and/or can bind to the surface of anHIV-infected cell. In certain aspects, at least two, at least three, orat least four antigen binding domains of the pentameric or hexameric HIVbinding molecule can specifically bind to an HIV virion particle, and/orcan bind to the surface of an HIV-infected cell.

An HIV antigen or epitope bound by a dimeric HIV binding moleculeprovided herein can be any one or more of the HIV proteins, includingthe HIV spike protein, e.g., gp120 and/or gp41, e.g., the immunodominantregion of gp41, the MPER, the CD4 binding site, the V1/V2 loop, the V3loop, and/or any carbohydrates associated with these regions. Moreover,a dimeric HIV binding molecule as provided herein can be multispecific,including antigen binding domains that specifically bind to two or moreantigens, e.g., one or more HIV antigens or epitopes and one or moreheterologous antigens or epitopes, or two or more HIV antigens orepitopes. For example in certain aspects a multispecific dimeric HIVbinding molecule provided herein can include two binding units eachcomprising two antigen binding domains, where at least two individualbinding domains bind to different antigens or epitopes. In certainaspects one or more binding domains can bind to, e.g., an epitope withinthe CD4 binding site of the spike protein, while one or more of theremaining binding domains can bind to, e.g., another epitope within theCD4 binding site, an epitope within the MPER region, any other region ofthe spike protein (exemplary binding domains are provided in Table 3),or an epitope of another HIV protein. In another aspect, a multispecificIgA binding molecule can comprise binding domains that are specific fordifferent subsets of or individual HIV groups or clades, therebyproviding a binding molecule with activity over a broader range of HIVviruses. Methods of making bispecific and multi-specific IgM and IgAbinding molecules, including antibodies, are described, for example, inPCT Publication No. WO 2015/053887 and PCT Publication No. WO2015/120474, the entire contents of which are hereby expresslyincorporated by reference. In certain aspects a heterologous bindingdomain can be associated with a J-chain, as described in PCT PublicationNo. WO 2015/153912, and elsewhere herein.

Multispecific Dimeric, Pentameric, or Hexameric HIV Binding Molecules

A multi-specific, e.g., bispecific dimeric HIV binding molecule asprovided herein can be based on the dimeric form of an IgA antibody or ahexameric or pentameric form of an IgM antibody, in which two, five, orsix pairs of heavy chain sequences can be present with or withoutassociated light chain sequences. For example, a bispecific dimeric HIVbinding molecule as provided herein can be composed of two IgA (IgA1 orIgA2) binding units, or five or six IgM binding units, and can include aJ-chain, e.g., a modified J-chain as provided elsewhere herein.

A multi-specific, e.g., bispecific dimeric HIV binding molecule asprovided herein can include mono- and/or bispecific binding units aslong as the molecule as a whole has at least two binding specificities,e.g., at least two non-identical antigen binding domains, e.g.,different regions of the gp120/41 spike protein, spike protein epitopesand epitopes from other HIV antigens, or HIV antigens and heterologousantigens. In certain aspects, one or more heterologous antigens can besituated on effector cells, e.g., CD3 on T-cells or CD16 on NK cells. Incertain aspects the non-identical antigen binding domain can be part ofa modified J-chain.

Thus, in one embodiment, a multi-specific, e.g., bispecific dimericbinding molecule as provided herein can include two monospecific bindingunits (AA, BB), each having bivalent binding specificity to a differentbinding target. In another embodiment, a multi-specific, e.g.,bispecific dimeric binding molecule as provided herein can include twobispecific binding units, each binding unit binding to the same twobinding targets (AB, AB) to form a bispecific dimeric binding molecule.In a further embodiment, one binding unit present in a multi-specificdimeric binding molecule as provided herein is monospecific (AA) whilethe other binding units are bispecific (BC), resulting in amultispecific binding molecule with three (A, B, C) bindingspecificities. In a further embodiment, each binding unit is bispecific,but one specificity is overlapping (e.g. AB, AC), resulting in amultispecific binding molecule with three (A, B, C) bindingspecificities. As discussed above for multispecific dimeric bindingmolecules, each of the five or six binding units can independently bemonospecific or bispecific (e.g., AA, BB, CC, etc.) or one or morebinding units can be bispecific (e.g., AB, AB, AC, CD, etc.). Thus, amulti-specific, e.g., bispecific pentameric or hexameric bindingmolecule as provided herein can include at least two independent antigenbinding domains, and up to twelve different, independent antigen bindingdomains. Other combinations, e.g., with four non-identical antigenbinding domains (A, B, C, and D) can be readily made based on thisdisclosure. In another embodiment all of the IgM or IgA binding unitscan be monospecific (e.g., AA) and the non-identical antigen bindingdomain can be part of a modified J-chain.

Modified J-Chains

In certain aspects, HIV binding molecules provided herein can bemultispecific, e.g., bispecific, incorporating a modified J-chain. Asprovided herein and in PCT Publication No. WO 2015/153912, a modifiedJ-chain can comprise a heterologous moiety, e.g., a heterologouspolypeptide, e.g., an additional desired binding domain, which caninclude, for example, a polypeptide binding domain capable ofspecifically binding to a target. The binding domain can be, forexample, an antibody or antigen binding fragment thereof, anantibody-drug conjugate or antigen binding fragment thereof, or anantibody-like molecule. A polypeptide binding domain can be introducedinto a J-chain by appropriately selecting the location and type ofaddition (e.g. direct or indirect fusion, chemical tethering, etc.).

In some embodiments, a modified J-chain can comprise a binding domainthat can include without limitation a polypeptide capable ofspecifically binding to a target antigen. In certain aspects, a bindingdomain associated with a modified J-chain can be an antibody or anantigen binding fragment thereof, including monospecific, bispecific,and multi-specific antibodies and antibody fragments. The antibodyfragment can be, without limitation, a Fab fragment, a Fab′ fragment, aF(ab′)₂ fragment, an scFv, (scFv)₂ fragment, single-chain antibodymolecules, single domain antibodies, e.g., camelid VHH antibodies,minibodies, or multispecific antibodies formed from antibody fragments.In certain aspects, the antibody fragment is a scFv.

In other aspects, the binding domain can be an antibody-like molecule,for example, a human domain antibody (dAb), Dual-Affinity Re-Targeting(DART) molecule, a diabody, a di-diabody, dual-variable domain antibody,a Stacked Variable Domain antibody, a Small Modular ImmunoPharmaceutical (SMIP), a Surrobody, a strand-exchange engineered domain(SEED)-body, or TandAb.

The binding domain can be introduced into the native J-chain sequence atany location that allows the binding of the binding domain to itsbinding target without interfering with the binding of the recipient IgMor IgA molecule to its binding target or binding targets or the abilityof the J-chain to effectively incorporate into an IgA dimer or an IgMpentamer. In certain aspects the binding domain can be inserted at ornear the C-terminus, at or near the mature N-terminus (i.e., amino acidnumber 23 of SEQ ID NO: 2 following cleavage of the signal peptide) orat an internal location that, based on the three-dimensional structureof the J-chain is accessible. In certain aspects, the binding domain canbe introduced into the native sequence J-chain without about 10 residuesfrom the C-terminus or without about 10 amino acid residues from themature N-terminus, of the human J-chain of SEQ ID NO: 2. In anotheraspect, the binding domain can be introduced into the native sequencehuman J-chain of SEQ ID NO: 2 in between cysteine residues 114 and 123of SEQ ID NO: 2, or at an equivalent location of another native sequenceJ-chain. In a further aspect, the binding domain can be introduced intoa native sequence J-chain, such as a J-chain of SEQ ID NO: 2, at or neara glycosylation site. In certain aspects, the binding domain can beintroduced into the native sequence human J-chain of SEQ ID NO: 2 withinabout 10 amino acid residues from the C-terminus.

Introduction can be accomplished by direct or indirect fusion, i.e. bythe combination of the J-chain and binding domain in one polypeptidechain by in-frame combination of their coding nucleotide sequences, withor without a peptide linker. The peptide linker (indirect fusion), ifused, can be about 1 to 50, or about 1 to 40, or about 1 to 30, or about1 to 20, or about 1 to 10, or about 1 to 5, or about 10 to 20 aminoacids in length, and can be present at one or both ends of the bindingdomain to be introduced into the J-chain sequence. In certain aspects,the peptide linker can be about 1 to 100 amino acids long. In certainaspects the peptide linker is 5, 10, 15, or 20 amino acids long.

In certain aspects the mature modified J-chain comprises the formulaX[L_(n)]J or J[L_(n)]X, where J is a native J-chain or functionalfragment thereof, e.g., a native human J-chain (amino acids 23 to 159 ofSEQ ID NO: 2), X is a binding domain, and [L_(n)] is a linker sequenceconsisting of n amino acids, where n is a positive integer, e.g., from 1to 100, 1 to 50, or 1 to 25. In certain aspects n=5, 10, 15, or 20. Incertain aspects L_(n) can consist of GGGGS (L₅, SEQ ID NO: 101),GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103),or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104). In certain aspects, X cancomprise an anti-CD3 binding domain, e.g., an anti-CD3 ScFv. In certainaspects X comprises or consists of SEQ ID NO: 105, SEQ ID NO: 106, orSEQ ID NO: 107.

It is also possible to introduce more than one heterologous polypeptide,e.g., more than one binding domain, into a J-chain.

The modified J-chain can be produced by well-known techniques ofrecombinant DNA technology, by expressing a nucleic acid encoding themodified J-chain in a suitable prokaryotic or eukaryotic host organism.

The modified J-chain can be co-expressed with the heavy and light chainsof the recipient IgM or IgA binding molecules as described elsewhereherein. The recipient binding molecule, prior to the modified J-chainincorporation can be monospecific, bispecific or multi-specific, e.g., amonospecific, bispecific, or multispecific IgA or IgM antibody.Bispecific and multi-specific IgM and IgA binding molecules, includingantibodies, are described, for example, in PCT Publication No. WO2015/053887 and PCT Publication No. WO 2015/120474, the entire contentsof which are hereby expressly incorporated by reference.

In certain aspects, an anti-HIV IgM or IgA binding molecule as describedherein can include a modified J-chain with binding specificity for animmune effector cell, such as a T-cell, NK-cell, a macrophage, or aneutrophil. In certain aspects the effector cell is a T-cell and thebinding target is CD3 (discussed below), or CD8. By activating andredirecting effector cells, e.g. effector T-cells (T-cell dependentkilling or TDCC), or NK cells to infected cells expressing HIV antigens,e.g., the HIV spike glycoprotein, on their surface, including reservoircells, a bispecific anti-HIV IgM or IgA binding molecule comprising aneffector cell-directed modified J-chain as provided herein can producean enhanced immune response against the target, the response comprising,e.g., complement-mediated cytotoxicity, antibody dependent cellularcytotoxicity (ADCC), TDCC, and/or NK-cell mediated killing, therebyfurther increasing potency and efficacy. In certain aspects, abispecific anti-HIV IgM or IgA binding molecule as provided hereincomprising a modified J-chain can be used for the treatment of a diseaseor condition caused by, or exacerbated by infection with HIV, and/or candirect HIV neutralization, and/or clearance or killing of anHIV-infected cells, such as reservoir cells.

In the case of T-cells, cluster of differentiation 3 (CD3) is amultimeric protein complex, known historically as the T3 complex, and iscomposed of four distinct polypeptide chains (ε, γ, δ, ζ) that assembleand function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complexserves as a T-cell co-receptor that associates non-covalently with theT-cell receptor (TCR). Components of this CD3 complex, especially CD3c,can be targets for a modified J-chain of a bispecific IgM or IgA bindingmolecule provided herein.

In certain aspects, a bispecific anti-HIV×anti-CD3 IgM or IgA bindingmolecule binds to HIV-infected cells or HIV virus particles via theantibody binding domains, while the J-chain is modified to bind to CD3,e.g., CD3ε.

In certain aspects the anti-CD3 binding domain of a modified J-chainprovided herein is a scFv. The anti CD3 scFv can be fused at or near theN-terminus of the J-chain, or at or near the C-terminus of the J-chaineither directly or indirectly via a synthetic linker introduced inbetween the scFv and the J-chain sequences, e.g., GGGGS (L₅, SEQ ID NO:101), GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO:103), or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104). Suitable anti-CD3binding domains for inclusion in a modified J-chain as provided hereininclude, but are not limited to, ScFv antigen binding domains comprisingthe amino acid sequences SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,as shown in Table 2.

TABLE 2 Exemplary CD3 Heterologous Binding Domains SEQ ID NO SourceBinds to Sequence 105 Kung, P.C., et CD3ϵQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRP al. (1979)GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL Science, 206,SSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSG 347-349GGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIK 106 U.S. Pat. No. CD3ϵQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAP 5,834,597GQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK 107 Beverley, P. C. CD3ϵEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPG & Canard, R. E.KGLEWVALINPYKGVTTYADSVKGRFTISVDKSKNTAYLQMN (1981) Eur. J.SLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG Immunol. 11,GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRN 329-334YLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK 112 GenBank: CD16EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPG ABQ52435.1KEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSS

In certain aspects the modified J-chain comprises the mature scFv aminoacid sequence of SEQ ID NO: 105 fused to the N-terminus of the humanJ-chain through a linker, e.g., GGGGS (L₅, SEQ ID NO: 101), GGGGSGGGGS(L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103), orGGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modified J-chain referredto herein as OL_(n)J, where n=5, 10, 15, or 20. OL_(n)J can furtherinclude a signal peptide to facilitate transport and assembly into anIgM or IgA binding molecules. In certain aspects the mature modifiedJ-chain comprises a scFv of SEQ ID NO: 105 fused to the C-terminus ofthe human J-chain through an amino acid linker, e.g., GGGGS (L₅, SEQ IDNO: 101), GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ IDNO: 103), or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modifiedJ-chain referred to herein as JL_(n)O, where n=5, 10, 15, or 20. JL_(n)Ocan further include a signal peptide, e.g., amino acids 1 to 22 of SEQID NO: 2, to facilitate transport and assembly into an IgM or IgAbinding molecules. In certain aspects, other signal peptides can beused. Selection and inclusion of suitable signal peptides to facilitateexpression, secretion, and incorporation of a modified J-chain into ananti-HIV IgM or IgA binding molecule as provided herein is well withinthe capabilities of a person of ordinary skill in the art.

In certain aspects the modified J-chain comprises the mature scFv aminoacid sequence SEQ ID NO: 106 fused to the N-terminus of the humanJ-chain through a linker, e.g., GGGGS (L₅, SEQ ID NO: 101), GGGGSGGGGS(L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103), orGGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modified J-chain referredto herein as VL_(n)J, where n=5, 10, 15, or 20. VL_(n)J can furtherinclude a signal peptide to facilitate transport and assembly into anIgM or IgA binding molecules. In certain aspects the mature modifiedJ-chain comprises a scFv of SEQ ID NO: 106 fused to the C-terminus ofthe human J-chain through an amino acid linker, e.g., GGGGS (L₅, SEQ IDNO: 101), GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ IDNO: 103), or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modifiedJ-chain referred to herein as JL_(n)V, where n=5, 10, 15, or 20. JL_(n)Vcan further include a signal peptide, e.g., amino acids 1 to 22 of SEQID NO: 2, to facilitate transport and assembly into an IgM or IgAbinding molecules. In certain aspects, other signal peptides can beused. Selection and inclusion of suitable signal peptides to facilitateexpression, secretion, and incorporation of a modified J-chain into ananti-HIV IgM or IgA binding molecule as provided herein is well withinthe capabilities of a person of ordinary skill in the art. Exemplarymodified J chains include, without limitation V5J (SEQ ID NO: 108), V10J(SEQ ID NO: 109), V15J (SEQ ID NO: 110), or V20J (SEQ ID NO: 111). Foreach sequence, the mature anti-CD3 ScFv sequence is single underlined,and the mature human J-chain sequence is shown in italics. In certainaspects, the mature modified J chain can SEQ ID NO: 108, SEQ ID NO: 109,SEQ ID NO: 110, SEQ ID NO: 111, or a combination thereof.

V5J (SEQ ID NO: 108) QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD. V10J (SEQ ID NO: 109)QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD. J-chain sequence for V15J(SEQ ID NO: 110) QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYP D.J-chain sequence for V20J (SEQ ID NO: 111)QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD.

In certain aspects the modified J-chain comprises the mature scFv aminoacid sequence SEQ ID NO: 107 fused to the N-terminus of the humanJ-chain through a linker, e.g., GGGGS (L₅, SEQ ID NO: 101), GGGGSGGGGS(L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103), orGGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modified J-chain referredto herein as UL_(n)J, where n=5, 10, 15, or 20. UL_(n)J can furtherinclude a signal peptide to facilitate transport and assembly into anIgM or IgA binding molecules. In certain aspects the mature modifiedJ-chain comprises a scFv of a SEQ ID NO: 107 fused to the C-terminus ofthe human J-chain through an amino acid linker, e.g., GGGGS (L₅, SEQ IDNO: 101), GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ IDNO: 103), or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modifiedJ-chain referred to herein as JL_(n)U, where n=5, 10, 15, or 20. JL_(n)Ucan further include a signal peptide, e.g., amino acids 1 to 22 of SEQID NO: 2, to facilitate transport and assembly into an IgM or IgAbinding molecules. In certain aspects, other signal peptides can beused. Selection and inclusion of suitable signal peptides to facilitateexpression, secretion, and incorporation of a modified J-chain into ananti-HIV IgM or IgA binding molecule as provided herein is well withinthe capabilities of a person of ordinary skill in the art.

A person of ordinary skill in the art would readily be able to makeadditional modified J-chains according to the description providedherein.

In certain aspects such as those noted above, a modified J-chainassociated with a dimeric or pentameric HIV binding molecule as providedherein can comprise an antigen binding domain that binds to an effectorcell, e.g. a T cell, an NK cell, or a macrophage. In certain aspects theeffector cell is a T cell, e.g., a cytotoxic T cell, expressing CD3,CD8, or a combination thereof. According to this aspect, the J-chain canbe modified by covalent attachment of a CD3 (CD3ε) binding domain or aCD8 binding domain. In this configuration, a dimeric or pentamericbinding molecule as provided herein provides binding specificity to atarget HIV antigen, e.g., an HIV spike protein, while the T-celltethering of the J-chain through CD3 binding or CD8 binding deliverscytotoxic potency. The CD3 binding domain or CD8 binding domaincovalently attached to a J-chain, or a variant thereof, can, for examplebe a single-chain Fv (scFv) of an anti-CD3 antibody, or a naturallyoccurring heavy chain only antibody, e.g. a camelid (camels, llamas,alpacas) or single-chain antibody of cartilaginous fish (sharks, rays),a scaffold, e.g. fibronectin (e.g. fibronectin III) with CD3 bindingspecificity.

In another aspect, the effector cell is a Natural killer (NK) cell. NKcells are important components of the innate immunity and play a keyrole in host defense by virtue of their ability to release cytokines andto mediate cytolytic activity against tumor cells and virus-infectedcells. NK cell antigens include, without limitation, CD16, CD32a, CD56,CD57, CD64, CD117 (or c-kit), adhesion molecules includinglymphocyte-associated molecule-2 (LFA-2 or CD2), LFA-3 (CD58), and LFA-1(CD11a/CD18). According to this aspect, the J-chain can be modified,e.g., by covalent attachment of a CD16 binding domain. In thisconfiguration, a dimeric or pentameric binding molecule as providedherein provides binding specificity to a target HIV antigen, e.g., anHIV spike protein, while the NK-cell tethering of the J-chain deliverscytotoxic potency. The CD16 binding domain covalently attached to aJ-chain, or a variant thereof can, for example be a single-chain Fv(scFv) of an anti-CD16 antibody, or a naturally occurring heavy chainonly antibody (VHH), e.g. a camelid (camels, llamas, alpacas) orsingle-chain antibody of cartilaginous fish (sharks, rays), a scaffold,e.g. fibronectin (e.g. fibronectin III) with CD16 binding specificity.

In certain aspects the modified J-chain comprises the mature VHH aminoacid sequence SEQ ID NO: 112 (Table 2) fused to the N-terminus of thehuman J-chain through a linker, e.g., GGGGS (L₅, SEQ ID NO: 101),GGGGSGGGGS (L₁₀, SEQ ID NO: 102), GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103),or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQ ID NO: 104), a modified J-chainreferred to herein as CD16L_(n)J, where n=5, 10, 15, or 20. CD16L_(n)Jcan further include a signal peptide to facilitate transport andassembly into an IgM or IgA binding molecules. An exemplary modifiedJ-chain according to this aspect can comprise the amino acid sequenceSEQ ID NO: 125, described in Example 1. In certain aspects the maturemodified J-chain comprises a VHH of SEQ ID NO: 112 fused to theC-terminus of the human J-chain through an amino acid linker, e.g.,GGGGS (L₅, SEQ ID NO: 101), GGGGSGGGGS (L₁₀, SEQ ID NO: 102),GGGGSGGGGSGGGGS (L₁₅, SEQ ID NO: 103), or GGGGSGGGGSGGGGSGGGGS (L₂₀, SEQID NO: 104), a modified J-chain referred to herein as JL_(n)CD16, wheren=5, 10, 15, or 20. JL_(n)CD16 can further include a signal peptide,e.g., amino acids 1 to 22 of SEQ ID NO: 2, to facilitate transport andassembly into an IgM or IgA binding molecules. In certain aspects, othersignal peptides can be used. Selection and inclusion of suitable signalpeptides to facilitate expression, secretion, and incorporation of amodified J-chain into an anti-HIV IgM or IgA binding molecule asprovided herein is well within the capabilities of a person of ordinaryskill in the art.

In another aspect, the effector cell is a macrophage. According to thisaspect, the J-chain can be modified, e.g., by covalent attachment of aCD14 binding domain. In this configuration, a dimeric or pentamericbinding molecule as provided herein provides binding specificity to atarget HIV antigen, e.g., an HIV spike protein, while the macrophagetethering of the J-chain delivers cytotoxic potency. The CD14 bindingdomain covalently attached to a J-chain, or a variant thereof can, forexample be a single-chain Fv (scFv) of an anti-CD14 antibody, or anaturally occurring heavy chain only antibody, e.g. a camelid (camels,llamas, alpacas) or single-chain antibody of cartilaginous fish (sharks,rays), a scaffold, e.g. fibronectin (e.g. fibronectin III) with CD14binding specificity.

In another aspect, the effector cell is a neutrophil. According to thisaspect, the J-chain can be modified, e.g., by covalent attachment of aCD16b or CD177 binding domain. In this configuration, a dimeric orpentameric binding molecule as provided herein provides bindingspecificity to a target HIV antigen, e.g., an HIV spike protein, whilethe neutrophil tethering of the J-chain delivers cytotoxic potency. TheCD16b or CD177 binding domain covalently attached to a J-chain, or avariant thereof can, for example be a single-chain Fv (scFv) of ananti-CD16b or CD177 antibody, or a naturally occurring heavy chain onlyantibody, e.g. a camelid (camels, llamas, alpacas) or single-chainantibody of cartilaginous fish (sharks, rays), a scaffold, e.g.fibronectin (e.g. fibronectin III) with CD16b or CD177 bindingspecificity.

Engineered HIV Antigen Binding Domains

In certain aspects an HIV antigen binding domain as provided herein caninclude as many as six immunoglobulin complementarity determiningregions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein at leastone, at least two, at least three, at least four, at least five, or atleast six CDRs are related to, or in some embodiments identical, oridentical except for one, two, three, four, five, six, seven, eight,nine, or ten single amino acid substitutions, to the corresponding CDRsof the HIV mAbs set forth in Table 3, below.

Methods for genetically engineering cloned variable regions intoimmunoglobulin domains, and expressing and purifying such constructs arepublished and within the capability of one of skill in the art. (See,for instance, Wu et al., MAbs, 1(4):339-47, 2009, and Wu et al., Nat.Biotechnol., 25:1290-7, 2007).

Exemplary peptide sequences encoding mature VH and VL domains thatcombine to form antibodies specific for HIV epitopes are set forth inTable 3. These sequences, or variants, fragments, or derivativesthereof, can then be engineered into a standard pentameric or hexamericIgM structure, as explained above, or an IgA structure.

TABLE 3 Exemplary Monoclonal Antibodies that Bind to HIV VH SEQ VL SEQReference Binding Site ID NO VH Sequence ID NO VL Sequence Wu et al.,CD4bs 5 QVQLVQSGGQMKKPGESMRISCRAS 6 EIVLTQSPGTLSLSPGETAIISCRTS Science,GYEFIDCTLNWIRLAPGKRPEWMGW QYGSLAWYQQRPGQAPRLVIYSG 329(5993): 856-LKPRGGAVNYARPLQGRVTMTRDVY STRAAGIPDRFSGSRWGPDYNLTI 861, 2010; WOSDTAFLELRSLTVDDTAVYFCTRGKN SNLESGDFGVYYCQQYEFFGQGT 2011/038290;CDYNWDFEHWGRGTPVIVSS KVQVDIKR U.S. Pat. No. CD4bs 7QVQLVQSGGQMKKPGESMRISCQAS 8 EIVLTQSPGTLSLSPGETAIISCRTS 8,637,036GYEFIDCTLNWVRLAPGRRPEWMGW QYGSLAWYQQRPGQAPRLVIYSGLKPRGGAVNYARPLQGRVTMTRDVY STRAAGIPDRFSGSRWGPDYNLTISDTAFLELRSLTADDTAVYYCTRGKN RNLESGDFGLYYCQQYEFFGQGT CDYNWDFEHWGRGTPVTVSSKVQVDIKR CD4bs 9 QVQLVQSGAVIKTPGSSVKISCRASGY 10EIVLTQSPGILSLSPGETATLFCKA NFRDYSIHWVRLIPDKGFEWIGWIKPLSQGGNAMTWYQKRRGQVPRLLI WGAVSYARQLQGRVSMTRQLSQDPD YDTSRRASGVPDRFVGSGSGTDFDPDWGVAYMEFSGLTPADTAEYFCV FLTINKLDREDFAVYYCQQFEFFGRRGSCDYCGDFPWQYWGQGTVVVV LGSELEVHR SS Wu et al., CD4bs 11QVQLVQSGSGVKKPGASVRVSCWTS 12 EIVLTQSPGTLSLSPGETASLSCTA Science,EDIFERTELIHWVRQAPGQGLEWIGW ASYGHMTWYQKKPGQPPKLLIFA 333(6049): 1593-VKTVTGAVNFGSPDFRQRVSLTRDRD TSKRASGIPDRFSGSQFGKQYTLT 1602, 2011LFTAHMDIRGLTQGDTATYFCARQKF ITRMEPEDFARYYCQQLEFFGQGYTGGQGWYFDLWGRGTLIVVSS TRLEIRR CD4bs 13 QVQLVQSGSGVKKPGASVRVSCWTS 14EIVLTQSPGTLSLSPGETASLSCTA EDIFERTELIHWVRQAPGQGLEWIGWASYGHMTWYQKKPGQPPKLLIFA VKTVTGAVNFGSPNFRHRVSLTRDRDTSKRASGIPDRFSGSQFGKQYTLT LFTAHMDIRGLTQGDTATYFCARQKFITRMEPEDFAGYYCQQVEFFGQG ERGGQGWYFDLWGRGTLIVVSS TRLEIR CD4bs 15QVQLVQSGAAVRKPGASVTVSCKFA 16 DIQMTQSPSSLSASLGDRVTITCQEDDDYSPYWVNPAPEHFIHFLRQAPG ASRGIGKDLNWYQQKAGKAPKLQQLEWLAWMNPTNGAVNYAWYLN LVSDASTLEGGVPSRFSGSGFHQGRVTATRDRSMTTAFLEVKSLRSDDT NFSLTISSLQAEDVATYFCQQYETAVYYCARAQKRGRSEWAYAHWGQG FGQGTKVDIK TPVVVSS CD4bs 17QVQLVQSGAAVRKPGASVTVSCKFA 18 DIQMTQSPSSLSASLGDRVTITCQEDDDYSPHWVNPAPEHYIHFLRQAPG ASRGIGKDLNWYQQKPGKAPKLQQLEWLAWMNPTNGAVNYAWQLH LVSDASILEGGVPSRFSGSGFHQNGRLTATRDGSMTTAFLEVRSLRSDDT FSLTISSLQPEDVATYFCQQYETFAVYYCARAQKRGRSEWAYAHWGQG GQGTKVDIK TPVAVSS CD4bs 19QVQLVQSGAAVRKPGASVTVSCKFA 20 DIQMTQSPSSLSASLGDRVTITCQEDDDFSPHWVNPAPEHYIHFLRQAPG ASRGIGKDLNWYQQKPGRAPKLQQLEWLAWMKPTNGAVNYAWQLQ LVSDASILEGGVPTRFSGSGFHQNGRVTVTRDRSQTTAFLEVKNLRSDDT FSLTISSLQAEDVATYFCQQYETFAVYYCARAQKRGRSEWAYAHWGQG GQGTKVDIK TPVVISA CD4bs 21QVQLVQSGAAVRKPGASISVSCKFAD 22 DIQMTQSPSSLSASLGDRVTITCQADDYSPHWMNPAPEHYIHFLRQAPG ASRGIGKDLNWYQQKRGRAPRL QQLEWLAWMNPTNGAVNYAWYLNLVSDASVLEGGVPSRFSGSGFHQ GRVTATRDRSMTTAFLEVRSLRSDDTNFSLTISTLQPEDVATYFCQQYET AVYYCARAQKRARSEWAYAHWGQG FGQGTKVDIK TPVVVSSCD4bs 23 QVQLVQSGAAVRKPGASVTVSCKFA 24 DIQMTQSPSSLSASLGDRVTITCQEDDDWSPHWVNPAPEHYIHFLRQAP ASRGIGKDLNWYQQKAGKAPKL GQQLEWLAWMNPTNGAVNYAWQLLVSDASILEGGVPSRFSGSGFHQN NGRLTATRDTSMTTAFLEVKSLRSDDFSLTISSLQPEDVATYFCQQYETF TAVYYCARAQKRGRSEWAYAHWGQ GQGTKVDIK GTPVVVSSLehman et al., V1/V2 25 QRLVESGGGVVQPGSSLRLSCAASGF 26QSALTQPASVSGSPGQSITISCNGT Science, DFSRQGMHWVRQAPGQGLEWVAFIKSNDVGGYESVSWYQQHPGKAPK 326(5950): 285- YDGSEKYHADSVWGRLSISRDNSKDTVVIYDVSKRPSGVSNRFSGSKSG 289, 2009 LYLQMNSLRVEDTATYFCVREAGGPNTASLTISGLQAEDEGDYYCKSL DYRNGYNYYDFYDGYYNYHYMDV TSTRRRVFGTGTKLTVLWGKGTTVTVSS V1/V2 27 QEQLVESGGGVVQPGGSLRLSCLASG 28QSALTQPASVSGSPGQTITISCNG FTFHKYGMHWVRQAPGKGLEWVALITSSDVGGFDSVSWYQQSPGKAPK SDDGMRKYHSDSMWGRVTISRDNSKVMVFDVSHRPSGISNRFSGSKSG NTLYLQFSSLKVEDTAMFFCAREAGGNTASLTISGLHIEDEGDYFCSSLT PIWHDDVKYYDFNDGYYNYHYMDV DRSHRIFGGGTKVTVLWGKGTTVTVSS Scheid et al., CD4bs 29 QVQLLQSGAAVTKPGASVRVSCEASG 30DIQMTQSPSSLSASVGDTVTITCQ Science, YNIRDYFIHWWRQAPGQGLQWVGWIANGYLNWYQQRRGKAPKLLIYD 333(6049): 1633- NPKTGQPNNPRQFQGRVSLTRHASWGSKLERGVPSRFSGRRWGQEYNL 1637, 2011 DFDTFSFYMDLKALRSDDTAVYFCARTINNLQPEDIATYFCQVYEFVVPG QRSDYWDFDVWGSGTQVTVSSASTK TRLDLKRTVAAP GP CD4bs31 QVRLSQSGGQMKKPGESMRLSCRAS 32 EIVLTQSPATLSLSPGETAIISCRTSGYEFLNCPINWIRLAPGRRPEWMGWL QSGSLAWYQQRPGQAPRLVIYSGKPRGGAVNYARKFQGRVTMTRDVYS STRAAGIPDRFSGSRWGADYNLSIDTAFLELRSLTSDDTAVYFCTRGKYC SNLESGDFGVYYCQQYEFFGQGTTARDYYNWDFEHWGRGAPVTVSSAS KVQVDIKRTVAAP TKGPSV Other 33QIHLVQSGTEVKKPGSSVTVSCKAYG 34 DIQMTQSPSTLSASIGDTVRISCRAVNTFGLYAVNWVRQAPGQSLEYIGQI SQSITGNWVAWYQQRPGKAPRLWRWKSSASHHFRGRVLISAVDLTGSS LIYRGAALLGGVPSRFSGSAAGTPPISSLEIKNLTSDDTAVYFCTTTSTYD DFTLTIGNLQAEDFGTFYCQQYDKWSGLHHDGVMAFSSWGQGTLISVS TYPGTFGQGTKVEVKRTVAAPSV AASTKGPSVFFIFPPSDEQLKSGT CD4bs 35 QVHLSQSGAAVTKPGASVRVSCEASG 36DIQMTQSPSSLSARVGDTVTITCQ YKISDHFIHWWRQAPGQGLQWVGWIANGYLNWYQQRRGKAPKLLIYD NPKTGQPNNPRQFQGRVSLTRQASW GSKLERGVPARFSGRRWGQEYNDFDTYSFYMDLKAVRSDDTAIYFCAR LTINNLQPEDVATYFCQVYEFIVPQRSDFWDFDVWGSGTQVTVSSASTK GTRLDLKRTVAA GPSX CD4bs 37SQHLVQSGTQVKKPGASVRVSCQAS 38 DIQMTQSPSSLSASVGDRVTINCQGYTFTNYILHWWRQAPGQGLEWMG AGQGIGSSLNWYQKKPGRAPKLLLIKPVFGAVNYARQFQGRIQLTRDIYR VHGASNLQRGVPSRFSGSGFHTTEIAFLDLSGLRSDDTAVYYCARDESG FTLTISSLQPDDVATYFCAVFQWFDDLKWHLHPWGQGTQVIVSPASTKG GPGTKVDIKRT P Walker et al., V3glycan 39QMQLQESGPGLVKPSETLSLTCSVSG 40 SDISVAPGETARISCGEKSLGSRA Nature,ASISDSYWSWIRRSPGKGLEWIGYVH VQWYQHRAGQAPSLIIYNNQDRP 477(7365)466-KSGDTNYSPSLKSRVNLSLDTSKNQV SGIPERFSGSPDSPFGTTATLTITS 70, 2011SLSLVAATAADSGKYYCARTLHGRRI VEAGDEADYYCHIWDSRVPTKWYGIVAFNEWFTYFYMDVWGNGTQVT VFGGGTTLTVL VSS V3glycan 41QVHLQESGPGLVKPSETLSLTCNVSG 42 TFVSVAPGQTARITCGEESLGSRSTLVRDNYWSWIRQPLGKQPEWIGYV VIWYQQRPGQAPSLIIYNNNDRPSHDSGDTNYNPSLKSRVHLSLDKSKNL GIPDRFSGSPGSTFGTTATLTITSVVSLRLTGVTAADSAIYYCATTKHGRR EAGDEADYYCHIWDSRRPTNWVIYGVVAFKEWFTYFYMDVWGKGTSV FGEGTTLIVL TVSS V3glycan 43QLHLQESGPGLVKPPETLSLTCSVSGA 44 SSMSVSPGETAKISCGKESIGSRASINDAYWSWIRQSPGKRPEWVGYVH VQWYQQKPGQPPSLIIYNNQDRPHSGDTNYNPSLKRRVTFSLDTAKNEV AGVPERFSASPDFRPGTTATLTITSLKLVDLTAADSATYFCARALHGKRI NVDAEDEADYYCHIYDARGGTNYGIVALGELFTYFYMDVWGKGTAVT WVFDRGTTLTVL VSS V3glycan 45QSQLQESGPRLVEASETLSLTCNVSGE 46 QSALTQPPSASGSPGQSITISCNGTSTGACTYFWGWVRQAPGKGLEWIGS ATNFVSWYQQFPDKAPKLIIFGVLSHCQSFWGSGWTFHNPSLKSRLTISL DKRPPGVPDRFSGSRSGTTASLTVDTPKNQVFLKLTSLTAADTATYYCAR SRLQTDDEAVYYCGSLVGNWDVFDGEVLVYNHWPKPAWVDLWGRGIP IFGGGTTLTVL VTVTVSS V3glycan 47QPQLQESGPGLVEASETLSLTCTVSGD 48 QSALTQPPSASGSPGQSISISCTGTSTAACDYFWGWVRQPPGKGLEWIGG SNRFVSWYQQHPGKAPKLVIYGVLSHCAGYYNTGWTYHNPSLKSRLTIS NKRPSGVPDRFSGSKSGNTASLTLDTPKNQVFLKLNSVTAADTAIYYCA VSGLQTDDEAVYYCSSLVGNWDRFDGEVLVYHDWPKPAWVDLWGRG VIFGGGTKLTVL TLVTVTVSS V3glycan 49QPQLQESGPGLVEASETLSLTCTVSGD 50 QSALTQPPSASGSPGQSITISCTGTSTGRCNYFWGWVRQPPGKGLEWIGS SNNFVSWYQQYPGKAPKLVIYEVLSHCRSYYNTDWTYHNPSLKSRLTISL NKRPSGVPDRFSGSKSGSTASLTVDTPKNQVFLRLTSVTAADTATYYCAR SGLQADDEGVYYCSSLVGNWDVFGGEVLVYRDWPKPAWVDLWGRGT IFGGGTKLTVL LVTVSS V3glycan 51QPQLQESGPTLVEASETLSLTCAVSGD 52 QSALTQPPSASGSPGQSITISCTGTSTAACNSFWGWVRQPPGKGLEWVGS SNNFVSWYQQHAGKAPKLVIYDLSHCASYWNRGWTYHNPSLKSRLTL VNKRPSGVPDRFSGSKSGNTASLALDTPKNLVFLKLNSVTAADTATYYC TVSGLQTDDEAVYYCGSLVGNWARFGGEVLRYTDWPKPAWVDLWGR DVIFGGGTKLTVL GTLVTVSS V3glycan 53QVQLQESGPGLVKPAETLSLTCSVSG 54 QSALTQPPSASGSLGQSVTISCNGESINTGHYYWGWVRQVPGKGLEWIG TSSDIGGWNFVSWYQQFPGRAPRHIHYTTAVLHNPSLKSRLTIKIYTLRN LIIFEVNKRPSGVPGRFSGSKSGNSQITLRLSNVTAADTAVYHCVRSGGDI ASLTVSGLQSDDEGQYFCSSLFGLYYYEWQKPHWFSPWGPGIHVTVSS RWDVVFGGGTKLTVL V3glycan 55QVQLQESGPGLVKPSETLSLTCTVSG 56 QSALTQPPSASGSLGQSLTISCSGTDSINTGHHYWGWVRQVPGKGPEWIA GSDIGSWNFVSWYQQFPGRAPNLHIHYNTAVLHNPALKSRVTISIFTLKN IIFEVNRRRSGVPDRFSGSKSGNTLITLSLSNVTAADTAVYFCVRSGGDIL ASLTVSGLRSEDEAEYFCSSLSGRYYIEWQKPHWFYPWGPGILVTVSS WDIVFGGGTKVTVL V3glycan 57QLQMQESGPGLVKPSETLSLSCTVSG 58 EIVMTQSPDTLSVSPGETVTLSCRDSIRGGEWGDKDYHWGWVRHSAGK ASQNINKNLAWYQYKPGQSPRLGLEWIGSIHWRGTTHYKESLRRRVSM VIFETYSKIAAFPARFVASGSGTEFSIDTSRNWFSLRLASVTAADTAVYFC TLTINNMQSEDVAVYYCQQYEEARHRHHDVFMLVPIAGWFDVWGPGV WPRTFGQGTKVDIK QVTVSS V1/V2 59QVQLVQSGPEVKKPGSSVKVSCKASG 60 DTVVTQSPLSLPVTPGEAASMSCSNTFSKYDVHWVRQATGQGLEWVGW STQSLRHSNGANYLAWYQHKPGMSHEGDKTESAQRFKGRVTFTRDTSA QSPRLLIRLGSQRASGVPDRFSGSSTAYMELRGLTSDDTAIYYCTRGSKH GSGTHFTLKISRVEAEDAAIYYCRLRDYVLYDDYGLINYQEWNDYLEF MQGLNRPWTFGKGTKLEIK LDVWGHGTAVTVSS V1/V2 61QVQLEQSGAEVKKPGSSVKVSCKASG 62 DTVVTQSPLSLPVTPGEAASMSCNTFSKYDVHWVRQATGQGLEWVGW TSTQSLRHSNGANYLAWYQHKPMSHEGDKTESAQRFKGRVTFTRDTSA GQSPRLLIRLGSQRASGVPDRFSGSTAYMELRGLTSDDTAIYYCTRGSKH SGSGTHFTLKISRVEPEDAAIYYCRLRDYVLYDDYGLINYQEWNDYLEF MQGLNRPWTFGKGTKLEIK LDVWGHGTAVTVSS V1/V2 63QVQLVQSGAEVKKPGSSVKVSCKAS 64 DTVVTQSPLSLSVTPGEAASMSCGNTFRKYDVHWVRQATGQGLEWVG TSTQSLRHSNGANYLAWYQHKPWMSHEGDKTESAQRFKGRVSFTRDN GQSPRLLIRLGSQRASGVPDRFSGSASTAYIELRGLTSDDTAIYYCTGGSK SGSGTHFTLKISRVEADDAAIYYCHRLRDYVLYDDYGLINQQEWNDYLE MQGLNRPWTFGKGTKLEIK FLDVWGHGTAVTVSS V1/V2 65QVQLVQSGAEVKKPGSSVKVSCKAS 66 EVVITQSPLFLPVTPGEAASLSCKGNSFSNHDVHWVRQATGQGLEWMG CSHSLQHSTGANYLAWYLQRPGWMSHEGDKTGLAQKFQGRVTITRDS QTPRLLIHLATHRASGVPDRFSGSGASTVYMELRGLTADDTAIYYCLTGS GSGTDFTLKISRVESDDVGTYYCKHRLRDYFLYNEYGPNYEEWGDYLA MQGLHSPWTFGQGTKVEIK TLDVWGHGTAVTVSS V1/V2 67QVQLVQSGPEVKKPGSSVKVSCKASG 68 DTVVTQSPLSLPVTPGEAASMSCSNTFSKYDVHWVRQATGQGLEWVGW STQSLRHSNGANYLAWYQHKPGISHERDKTESAQRFKGRVTFTRDTSAT QSPRLLIRLGSQRASGVPDRFSGSTAYMELRGLTSDDTAIYYCTRGSKHR GSGTHFTLKISRVEAEDAAIYYCLRDYVLYDDYGLINYQEWNDYLEFL MQGLNRPWTFGKGTKLEIK DVWGHGTAVTVSSZhu et al., J. MPER 69 QVQLVQSGAEVKKPGESLKISCKVSG 70DIQLTQSPSSLSASLGDKVTITCR Virol., YNFASEWIGWVRQMPGKGLEWMGIIASQHIKKYLNWYQQKPGKAPKL 85(21): 11401- YPGDSDTKYSPSFQGQVIISADKSINTLIYGALNLQSGVPSRFSGRGSGTD 11408, 2011 AYLQWSSLKASDTAIYYCARQNHYGFTLTISSLQPEDFATYYCQQSYST SGSYFYRTAYYYAMDVWGQGTTVT PFTFGPGTKVDIKR VSSLiao et al., CD4bs 71 SETLSLTCTVSGGSMGGTYWSWLRLS 72SYELTQPPSVSVSPGQTATITCSG Nature, PGKGLEWIGYIFHTGETNYSPSLKGRVASTNVCWYQVKPGQSPEVVIFEN 496(7446): 469- SISVDTSEDQFSLRLRSVTAADTAVYFYKRPSGIPDRFSGSKSGSTATLTIR 476, 2013 CASLPRGQLVNAYFRNWGRGSLVSVGTQAIDEADYYCQVWDSFSTFVF TA GSGTQVTVL Huang e tal., MPER 73EVQLVESGGGLVKPGGSLRLSCSASG 74 SYELTQETGVSVALGRTVTITCRG Nature,FDFDNAWMTWVRQPPGKGLEWVGRI DSLRSHYASWYQKKPGQAPILLF 491(7424): 406-TGPGEGWSVDYAAPVEGRFTISRLNSI YGKNNRPSGVPDRFSGSASGNRA 412, 2012;NFLYLEMNNLRMEDSGLYFCARTGK SLTISGAQAEDDAEYYCSSRDKS WOYYDFWSGYPPGEEYFQDWGRGTLVT GSRLSVFGGGTKLTVLX 2013/070776 VSS WO CD4bs 75QVRLSQSGGQMKKPGDSMRISCRASG 6 EIVLTQSPGTLSLSPGETAIISCRTS 2013/086533YEFINCPINWIRLAPGKRPEWMGWM QYGSLAWYQQRPGQAPRLVIYSGKPRGGAVSYARQLQGRVTMTRDMYS STRAAGIPDRFSGSRWGPDYNLTIETAFLELRSLTSDDTAVYFCTRGKYC SNLESGDFGVYYCQQYEFFGQGTTARDYYNWDFEHWGQGTPVTVSS KVQVDIKR WO CD4bs/b19 77QVQLVQPGTAMKSLGSSLTITCRVSG 78 QSALTQPASVSGSPGQSINISCAG 2013/142324DDLGSFHFGTYFMIWVRQAPGQGLE RSDRVSWYQQRPNGVPKLLMFDYMGGILPSTKTPTYAHKFRGRVSISAP VYRRPSGVSDRFSGSHSGDTAFLGVPPVLSLALTNLTYDDTATYFCARE TISGLQTEDEADYYCTSHPYAFGRGRHFEPKNRDNLEGKFFDLWGRGTF AGTKVNVL VRVSP Diskin et al.,J. CD4bs 79XVRLSQSGGQMKKPGESMRLSCRAS 80 EIVLTQSPATLSLSPGETAIISCRTS Exp. Med.,GYEFLNCPINWIRLAPGRRPEWMGWL QYGSLAWYQQRPGQAPRLVIYSG 210(6): 1235-KPRWGAVNYARKFQGRVTMTRDVY STRAAGIPDRFSGSRWGADYNLSI 1249, 2013SDTAFLELRSLTSDDTAVYFCTRGKY SNLESGDFGVYYCQQYEFFGQGTCTARDYYNWDFEHWGRGAPVTVSS KVQVDIKR Mouquet et al., V3glycan 81QVQLQESGPGLVKPSETLSVTCSVSG 82 SYVRPLSVALGETARISCGRQAL PNAS USA,DSMNNYYWTWIRQSPGKGLEWIGYIS GSRAVQWYQHRPGQAPILLIYNN 109: E3268-DRESATYNPSLNSRVVISRDTSKNQLS QDRPSGIPERFSGTPDINFGTRATL E3277, 2012LKLNSVTPADTAVYYCATARRGQRIY TISGVEAGDEADYYCHMWDSRSGVVSFGEFFYYYSMDVWGKGTTVTV GFSWSFGGATRLTVLG SS Huang, J. et al. face of83 QGQLVQSGAELKKPGASVKISCKTSG 84 QSVLTQSASVSGSLGQSVTISCTG Nature 515,contiguous YRFNFYHINWIRQTAGRGPEWMGWIS PNSVCCSHKSISWYQWPPGRAPT138-142 (2014) areas of gp41 PYSGDKNLAPAFQDRVIMTTDTEVPVLIIYEDNERAPGISPRFSGYKSYW and gp120 TSFTSTGAAYMEIRNLKFDDTGTYFCSAYLTISDLRPEDETTYYCCSYTH AKGLLRDGSSTWLPYLWGQGTLLTV NSGCVFGTGTKVSVL SSBonsignori M, V1/V2 85 EVQLVESGANVVRPGGSLRLSCKASG 86EIVLAQSPGTLSLSPGERATLSCR et al., J Virol. FIFENFGFSWVRQAPGKGLQWVAGLASHNVHPKYFAWYQQKPGQSPR 85(19): 9998- NWNGGDTRYADSVKGRFRMSRDNSRLLIYGGSTRAAGIPGKFSGSGSGT 10009 2011. NFVYLDMDKVGVDDTAFYYCARGTDFTLTISRVDPEDFAVYYCQQYG DYTIDDAGIHYQGSGTFWYFDLWGR GSPYTFGQGTKVEIKGTLVSVSS V1/V2 87 EVQLVESGGSVVRPGGSLRLSCRASG 88 EIVLTQSPATLSVSPGERATLSCRFIFENYGLTWVRQVPGKGLHWVSGM ASQNVHPRYFAWYQQKRGQSPRNWNGGDTRYADSVRGRFSMSRDNSN LLIHSGSTRAAGIADRFSGGGSGMNIAYLQMNNLRVEDTALYYCARGTD HFTLTITRVEPEDFAVYFCQQYGYTIDDQGRFYQGSGTFWYFDFWGRG GSPYTFGQGTRVELR TLVTVSS V1/V2 89EVQLVESGGGVVRPGGSLRLSCAASG 90 EIVLTQSPATLSLSPGERATLSCRFIFENYGLTWVRQVPGKGLHWVSGM ASQSVHPKYFAWYQQKPGQSPRNWNGGDTRYADSVRGRFSMSRDNSN LLIYSGSTRAAGIADRFSGGGSGINIAYLQMKNLRVDDTALYYCARGTD HFTLTITRVEPEDFAVYFCQQYGYTIDDQGIFYKGSGTFWYFDLWGRGT GSPYTFGQGTKVELR LVTVSS V1/V2 91EVQLVESGGGLIRPGGSLRLSCKGSGF 92 EIVLTQSPDTLSLSPGERATLSCRIFENFGFGWVRQGPGKGLEWVSGTN ASQSVHSRYFAWYQHKPGQPPRLWNGGDSRYGDSVKGRFTISRDNSNNF LIYGGSTRATGIPNRFSAGGSGTQVYLQMNSLRPEDTAIYYCARGTDYTI FTLTVNRLEAEDFAVYYCQQYGRDDQGIRYQGSGTFWYFDVWGRGTLV SPYTFGQGTKVEIR TVSS Sok, D. et al., 93QVHLTQSGPEVRKPGTSVKVSCKAPG DFVLTQSPHSLSVTPGESASISCKS Proc. Natl.NTLKTYDLHWVRSVPGQGLQWMGW SHSLIHGDRNNYLAWYVQKPGRS Acad. Sci. USAISHEGDKKVIVERFKAKVTIDWDRST PQLLIYLASSRASGVPDRFSGSGS 111: 17624-NTAYLQLSGLTSGDTAVYYCAKGSK DKDFTLKISRVETEDVGTYYCMQ 17629(2014)HRLRDYALYDDDGALNWAVDVDYL GRESPWTFGQGTKVDIK SNLEFWGQGTAVTVSSBuchacher, A., V3/glycan 95 EVQLVESGGGLVKAGGSLILSCGVSN 96DIQMTQSPSTLSASVGDTITITCRA et al., AIDS FRISAHTMNWVRRVPGGGLEWVASISSQSIETWLAWYQQKPGKAPKLLI Res. Hum. TSSTYRDYADAVKGRFTVSRDDLEDFYKASTLKTGVPSRFSGSGSGTEFT Retroviruses VYLQMHKMRVEDTAIYYCARKGSDRLTISGLQFDDFATYHCQHYAGYS 10: 359-369 LSDNDPFDAWGPGTVVTVSP ATFGQGTRVEIK(1994); WO 2011/035205 Pincus SH, et The immuno- 97QVQLVQSGGGVFKPGGSLRLSCEASG 98 DIVMTQSPDSLAVSPGERATIHCK al. J Immunoldominant FTFTEYYMTWVRQAPGKGLEWLAYI SSQTLLYSSNNRHSIAWYQQRPG 170: 2236-region of gp41 SKNGEYSKYSPSSNGRFTISRDNAKNS QPPKLLLYWASMRLSGVPDRFSG2241(2003) VFLQLDRLSADDTAVYYCARADGLT SGSGTDFTLTINNLQAEDVAIYYCYFSELLQYIFDLWGQGARVTVSS HQYSSHPPTFGHGTRVEIK Moore, J. P., CD4bs 99QVQLQESGPGLVKPSQTLSLSCTVSG 100 QSVLTQPPSASGSPGQSVTISCTG and J. SodroskiGSSSSGAHYWSWIRQYPGKGLEWIGY TSSDVGGYNYVSWYQHHPGKAP J. VirolIHYSGNTYYNPSLKSRITISQHTSENQF KLIISEVNNRPSGVPDRFSGSKSG 70: 1863-1872SLKLNSVTVADTAVYYCARGTRLRTL NTASLTVSGLQAEDEAEYYCSSY (1996); WORNAFDIWGQGTMVTVSS TDIHNFVFGGGTKLTVLR 2006/044410

In certain aspects the HIV antigen binding domain of a dimeric,hexameric, or pentameric binding molecule as provided herein comprisesan antibody heavy chain variable region (VH) and an antibody light chainvariable region (VL), wherein the VH region, the VL region, or both theVH and VL regions are related to the corresponding VH and VL of HIVmonoclonal antibodies disclosed in the references set forth in Table 3,above. In certain aspects, the binding molecules provided herein exhibitgreater potency than an IgG antibody comprising the VH and VL ofantibodies listed in Table 3.

In certain aspects the VH can comprise an amino acid sequence at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or 100% identical to any one or more ofthe amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, or SEQ ID NO: 99.

In certain aspects the VL can comprise an amino acid sequence at leastat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or 100% identical to any one ormore of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 6, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ IDNO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, orSEQ ID NO: 100.

In certain aspects the VH and VL amino acid sequences can comprise aminoacid sequences at least at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or100% identical to SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ IDNO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12,SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ IDNO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 andSEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ IDNO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO:34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38,SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 andSEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 55 and SEQ IDNO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO:60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64,SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ IDNO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 6, SEQ ID NO: 77 and SEQID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO:82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86,SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ IDNO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, SEQ ID NO: 95and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, or SEQ ID NO: 99 andSEQ ID NO: 100, respectively.

In certain aspects the HIV antigen binding domain of a dimeric,hexameric, or pentameric binding molecule as provided herein comprisesthe HCDR1, HCDR2, and HCDR3 regions, or HCDR1, HCDR2, and HCDR3 regionscontaining one or two single amino acid substitutions, and the LCDR1,LCDR2, and LCDR3 regions, or LCDR1, LCDR2, and LCDR3 containing one ortwo single amino acid substitutions, of the VH and VL amino acidsequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 andSEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ IDNO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34,SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ IDNO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 43and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 andSEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ IDNO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO:56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60,SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ IDNO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 andSEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 6, SEQ ID NO: 77 and SEQ IDNO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO:82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86,SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ IDNO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, SEQ ID NO: 95and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, or SEQ ID NO: 99 andSEQ ID NO: 100.

In certain aspects a hexameric or pentameric IgM antibody designatedherein as HIV02M is provided comprising an IgM heavy chain comprisingthe amino acid sequence SEQ ID NO: 114 and a kappa light chaincomprising the amino acid sequence SEQ ID NO: 115. In certain aspects ahexameric or pentameric IgM antibody designated herein as HIV12M isprovided comprising an IgM heavy chain comprising the amino acidsequence SEQ ID NO: 117 and a kappa light chain comprising the aminoacid sequence SEQ ID NO: 118. In certain aspects a hexameric orpentameric IgM antibody designated herein as HIV32M is providedcomprising an IgM heavy chain comprising the amino acid sequence SEQ IDNO: 120 and a kappa light chain comprising the amino acid sequence SEQID NO: 121. In certain aspects a hexameric or pentameric IgM antibodydesignated herein as HIV72M is provided comprising an IgM heavy chaincomprising the amino acid sequence SEQ ID NO: 123 and a kappa lightchain comprising the amino acid sequence SEQ ID NO: 124. Where the IgMantibody HIV02M, HIV12M, HIV32M, or HIV72M is pentameric, it can furtherinclude a J-chain or functional fragment thereof, e.g., a wild-typehuman J-chain comprising amino acids 23 to 159 of SEQ ID NO: 2, or amodified J-chain as provided elsewhere herein, e.g., a J-chaincomprising the formula X[L_(n)]J or J[L_(n)]X, where J is a nativeJ-chain or functional fragment thereof, e.g., a native human J-chain(amino acids 23 to 159 of SEQ ID NO: 2), X is a binding domain, e.g.,SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 112, and[L_(n)] is a linker sequence consisting of n amino acids, where n is apositive integer, e.g., from 1 to 100, 1 to 50, or 1 to 25. In certainaspects n=5, 10, 15, or 20, e.g., SEQ ID NO: 101, SEQ ID NO: 102, SEQ IDNO: 103, or SEQ ID NO: 104. In certain aspects the modified J-chain cancomprise or consist of V5J (SEQ ID NO: 108), V10J (SEQ ID NO: 109), V15J(SEQ ID NO: 110), V20J (SEQ ID NO: 111), C15J (SEQ ID NO: 125), or anycombination thereof.

In certain aspects an IgM antibody as provided herein, e.g., an IgMantibody comprising HIV02M, HIV12M, HIV32M, or HIV72M, either without aJ-chain or further comprising a wild-type or modified J-chain asprovided herein can exhibit greater potency than a single binding unitantibody, e.g., an IgG antibody comprising the corresponding VH and VLof an antibody listed in Table 3. For example, an IgM antibody asprovided herein, e.g., an IgM antibody comprising HIV02M, HIV12M,HIV32M, or HIV72M can more potently neutralize HIV, bind and neutralizemore diverse HIV variants or clades, enhance viral clearance, and/or bemore potent in preventing, controlling or treating HIV infection than acorresponding reference single binding unit molecule comprising only twoHIV antigen binding domains. Moreover, an IgM antibody as providedherein, e.g., an IgM antibody comprising HIV02M, HIV12M, HIV32M, orHIV72M can be more potent in controlling HIV infectivity and growth ascompared with a corresponding reference single binding unit moleculecomprising only two HIV antigen binding domains. In addition, an IgMantibody as provided herein, e.g., an IgM antibody comprising HIV02M,HIV12M, HIV32M, or HIV72M can be used to treat chronic infection, e.g.,by binding to and/or effecting antibody and/or cell-mediated killing ofHIV infected cells, e.g., reservoir cells that express extremely lowlevels of HIV antigens on their surface. In a further example, an IgMantibody as provided herein, e.g., an IgM antibody comprising HIV02M,HIV12M, HIV32M, or HIV72M can be more effective at activating andkilling such HIV-infected cells or killing such cells after activationwith an independent activating agent such as an effector cell, e.g., aT-cell. In a further example, an IgM antibody as provided herein, e.g.,an IgM antibody comprising HIV02M, HIV12M, HIV32M, or HIV72M can provideequivalent benefit at a lower dosage than that of a correspondingreference single binding unit molecule comprising only two HIV antigenbinding domains. In certain aspects, administration of an IgM antibodyas provided herein, e.g., an IgM antibody comprising HIV02M, HIV12M,HIV32M, or HIV72M can allow for reduced or modified dosages of otheranti-retroviral therapies, such as ART (see, e.g., Example 7 below).

While a variety of different dimeric, hexameric, and pentameric bindingmolecules can be contemplated by a person of ordinary skill in the artbased on this disclosure, and as such are included in this disclosure,in certain aspects, a binding molecule as described above is provided inwhich each binding unit comprises two IgM heavy chains each comprising aVH situated amino terminal to the IgM constant region or fragmentthereof, and two immunoglobulin light chains each comprising a VLsituated amino terminal to an immunoglobulin light chain constantregion. In certain aspects, a binding molecule as described above isprovided in which each binding unit comprises two IgA heavy chains eachcomprising a VH situated amino terminal to the IgA constant region orfragment thereof, and two immunoglobulin light chains each comprising aVL situated amino terminal to an immunoglobulin light chain constantregion.

Moreover in certain aspects, at least one binding unit of the bindingmolecule, or two, three, four, five, or six binding units of the bindingmolecule, each comprise two of the HIV antigen binding domains asdescribed above. In certain aspects the two HIV antigen binding domainsin the one binding unit of the binding molecule, or two, three, four,five, or six binding units of the binding molecule, can be differentfrom each other, or they can be similar or identical.

In certain aspects, the two IgA heavy chains within one binding unit ofthe binding molecule, or two binding units of the binding molecule, areidentical. In certain aspects, the two IgM heavy chains within onebinding unit of the binding molecule, or two, three, four, five, or sixbinding units of the binding molecule, are identical.

In certain aspects, the two light chains within one binding unit of thebinding molecule, or two, three, four, five, or six binding units of thebinding molecule, are identical. In certain aspects, two identical lightchains within at least one binding unit, or within two, three, four,five, or six binding units of the binding molecule are kappa lightchains, e.g., human kappa light chains, or lambda light chains, e.g.,human lambda light chains.

In certain aspects at least one, two, three, four, five, or six bindingunits of a dimeric, pentameric, or hexameric HIV binding molecule, e.g.,an IgM antibody provided by this disclosure comprises or each comprisetwo identical IgA or IgM heavy chains, and two identical light chains.According to this aspect, the HIV antigen binding domains in the onebinding unit of the binding molecule, or two, three, four, five, or sixbinding units of the binding molecule, can be identical. Furtheraccording to this aspect, a dimeric, pentameric, or hexameric HIVbinding molecule, e.g., an IgM antibody as provided herein can compriseat least one, two, three, four, five, six, seven, eight, nine, ten,eleven, or twelve copies of an HIV antigen binding domain as describedabove. In certain aspects at least two, at least three, at least four,at least five, or at least six of the binding units can be identicaland, in certain aspects the binding units can comprise identical antigenbinding domains, e.g., at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten, at least eleven, or at least twelve HIV antigen bindingdomains can be identical.

In certain aspects, a dimeric, pentameric, or hexameric HIV bindingmolecule as provided herein can possess advantageous structural and/orfunctional properties, or “improved binding characteristics,” ascompared to other binding molecules, such as a corresponding referencesingle binding unit molecule comprising the same antigen binding domain.For example, the dimeric, pentameric, or hexameric HIV binding moleculecan possess improved activity or potency in a biological assay, eitherin vitro or in vivo, relative to a corresponding reference singlebinding unit molecule, e.g., an IgG1 binding molecule comprising thesame VH and VL region sequences as are present in the multimeric bindingmolecule, as described above. Biological assays include, but are notlimited to, Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) assays,T-cell dependent Cellular Cytotoxicity (TDCC) assays,Complement-Dependent Cytotoxicity (CDC) assays, Cell-To-Cell Spread(CTCS) assays, viral transcytosis assays, complement-dependent virolysisassays, virus neutralization assays, cell attachment assays, viralegress assays, immunohistochemical assays, direct cytotoxicity assays,complement-mediated cytotoxicity assays, etc. Suitable HIVglycoprotein-expressing cells for performing such assays include, butare not limited to .g., CHO-gp120, CHO-gp140, Jurkat-522 F/Y cells, ormammalian cells expressing membrane anchored trimeric forms of gp140,e.g., strain JR-FL (Go et al. 2015 J Virol 89:8245-8257). In certainaspects a dimeric, pentameric, or hexameric HIV binding molecule, e.g.,an IgM antibody as provided herein can direct HIV neutralization, and/orclearance or killing of an HIV-infected cells, such as reservoir cells,at higher potency than an equivalent amount of a monospecific, bivalentIgG1 antibody or fragment thereof that specifically binds to the sameHIV epitope as the HIV antigen binding domain.

By “potency” or “binding characteristics” refers to the ability of abinding molecule to achieve a given biological result. For example,potency can be referred to as the amount of a given binding moleculenecessary to achieve a given biological result (EC100 or IC100) or theamount of a given binding molecule necessary to achieve 50% of a desiredbiological result (EC50 or IC50). Biological results can include, forexample, binding to recombinant gp120 or gp41, binding to gp120/41expressing cells, binding to chronically and/or latently infected celllines, neutralization of HIV viruses or HIV pseudo-typed viruses,neutralization of more diverse HIV viruses or HIV pseudo-typed viruses,killing of latent HIV-infected cells, reduction of HIV virus or HIVinfected cells in therapeutic animal models, or prolonged absence of HIVvirus after cessation of ART.

Polynucleotides, Vectors, and Host Cells

The disclosure further provides a polynucleotide, e.g., an isolated,recombinant, and/or non-naturally-occurring polynucleotide, comprising anucleic acid sequence that encodes a polypeptide subunit of the dimeric,hexameric, or pentameric binding molecule as described above. By“polypeptide subunit” is meant a portion of a binding molecule, bindingunit, or antigen binding domain that can be independently translated.Examples include, without limitation, an antibody variable domain, e.g.,a VH or a VL, a J-chain, a secretory component, a single chain Fv, anantibody heavy chain, an antibody light chain, an antibody heavy chainconstant region, an antibody light chain constant region, and/or anyfragment, variant, or derivative thereof.

In certain aspects, the polypeptide subunit can comprise an IgM or anIgA heavy chain constant region or fragment thereof, and VH portion ofan HIV antigen binding domain. In certain aspects the polynucleotide canencode a polypeptide subunit comprising a human IgM or IgA constantregion or fragment thereof fused to the C-terminal end of a VH, wherethe VH comprises the HCDR1, HCDR2, and HCDR3 regions, or the HCDR1,HCDR2, and HCDR3 regions containing one or two single amino acidsubstitutions of a VH comprising the amino acid sequence SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ IDNO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99.

In certain aspects, the polypeptide subunit can comprise an antibody VLportion of an HIV antigen binding domain as described above. In certainaspects the polypeptide subunit can comprise a human antibody lightchain constant region or fragment thereof fused to the C-terminal end ofa VL, where the VL comprises LCDR1, LCDR2, and LCDR3 regions, or theLCDR1, LCDR2, and LCDR3 regions containing one or two single amino acidsubstitutions of a VL comprising the amino acid sequence SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 6, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100.

In certain aspects the polynucleotide can encode a polypeptide subunitcomprising a human IgM or IgA constant region or fragment thereof fusedto the C-terminal end of a VH, where the VH comprises an amino acidsequence at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or 100% identical toany one or more of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83,SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO:93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99.

In certain aspects the polynucleotide can encode a polypeptide subunitcomprising a human light chain constant region or fragment thereof fusedto the C-terminal end of a VL, where the VL comprises an amino acidsequence at least at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to any one or more of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 6,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ IDNO: 96, SEQ ID NO: 98, or SEQ ID NO: 100.

Thus, to form the antigen binding domains, the variable regions ofantibodies that specifically bind to an HIV antigen, e.g., thatspecifically bind to an epitope on the HIV spike protein, can beinserted into expression vector templates for IgM and/or IgA structures,thereby creating multimeric binding molecules having at least twobivalent binding units. In brief, nucleic acid sequences encoding theheavy and light chain variable domain sequences can be synthesized oramplified from existing molecules, and inserted into vectors in theproper orientation and in frame such that upon expression, the vectorwill yield a full length heavy or light chain. Vectors useful for thesepurposes are known in the art. Such vectors can also comprise enhancerand other sequences needed to achieve expression of the desired chains.Multiple vectors or single vectors can be used. These vectors aretransfected into host cells and then the chains are expressed andpurified. Upon expression the chains form fully functional multimericbinding molecules, as has been reported in the literature. The fullyassembled multimeric binding molecules can then be purified by standardmethods. The expression and purification processes can be performed atcommercial scale, if needed.

The disclosure further provides a composition comprising two or morepolynucleotides, where the two or more polynucleotides collectively canencode a dimeric, hexameric, or pentameric binding molecule as describedabove. In certain aspects the composition can include a polynucleotideencoding an IgM and/or IgA heavy chain or fragment thereof, e.g., ahuman IgM heavy chain as described above where the IgM and/or IgA heavychain comprises at least the VH of an HIV antigen binding domain, and apolynucleotide encoding a light chain or fragment thereof, e.g., a humankappa or lambda light chain that comprises at least the VL of an HIVantigen binding domain. A polynucleotide composition as provided canfurther include a polynucleotide encoding a J-chain, e.g., a humanJ-chain, or a fragment, variant, or derivative thereof. In certainaspects the polynucleotides making up a composition as provided hereincan be situated on two, three, or more separate vectors, e.g.,expression vectors. Such vectors are provided by the disclosure. Incertain aspects two or more of the polynucleotides making up acomposition as provided herein can be situated on a single vector, e.g.,an expression vector. Such a vector is provided by the disclosure.

The disclosure further provides a host cell, e.g., a prokaryotic oreukaryotic host cell, comprising a polynucleotide or two or morepolynucleotides encoding a dimeric, pentameric, or hexameric HIV bindingmolecule as provided herein, or any subunit thereof, a polynucleotidecomposition as provided herein, or a vector or two, three, or morevectors that collectively encode a dimeric, pentameric, or hexameric HIVbinding molecule as provided herein, or any subunit thereof. In certainaspects a host cell provided by the disclosure can express a dimeric,pentameric, or hexameric HIV binding molecule as provided by thisdisclosure, or a subunit thereof.

In a related aspect, the disclosure provides a method of producing adimeric, pentameric, or hexameric HIV binding molecule as provided bythis disclosure, where the method comprises culturing a host cell asdescribed above, and recovering the binding molecule.

Methods of Use

This disclosure provides improved methods for preventing, controlling,or treating HIV infection, and/or methods for neutralizing HIVinfectivity, e.g., across two or more types, groups, or clades, using adimeric IgA-based HIV binding molecule, or pentameric or hexamericIgM-based HIV binding molecule. The methods described below can utilizemultimeric binding molecules comprising HIV antigen binding domainsincluding without limitation, the antibodies and corresponding VH and VLsequences disclosed in the references set forth in Table 3, or variants,derivatives, or analogs thereof, where the dimeric, pentameric, orhexameric HIV binding molecule can provide improved virus neutralizationand/or clearance potency as compared to a corresponding reference singlebinding unit molecule, fragment, variant, derivative, or analog, asdisclosed and explained above. Exemplary corresponding single bindingunit molecules are described in the references presented in Table 3.Based on this disclosure, construction of a dimeric IgA bindingmolecule, or pentameric or hexameric IgM binding molecule comprising anyHIV-specific antigen binding domain of interest is well within thecapabilities of a person of ordinary skill in the art. The improvedbinding characteristics of such compositions can, for example, allow areduced dose to be used, or can result in more effective neutralizationof viruses resistant to neutralization by the original antibody, asexplained above. By “resistant” is meant any degree of reduced activityof an HIV antibody, on HIV infectivity, replication, release, etc.

In certain aspects, this disclosure provides a method for directingimproved neutralization of HIV, or killing of HIV infected cells, wherethe method includes contacting an HIV, or an HIV-infected cell with adimeric, pentameric, or hexameric HIV binding molecule, e.g., an IgMantibody as described herein, where the binding molecule can directvirus neutralization, or killing of HIV reservoir cells, at a higherpotency than an equivalent amount of a corresponding reference singlebinding unit molecule, e.g., a monospecific, bivalent IgG antibody orfragment thereof. In certain aspects a dimeric, pentameric, or hexamericHIV binding molecule, e.g., an IgM antibody as provided herein candirect virus neutralization of two or more HIV types, subtypes or cladesat higher potency than an equivalent amount of a corresponding referencesingle binding unit molecule, where the corresponding reference singlebinding unit molecule is, or comprises similar or identical VH and VLregions as at least one binding unit of a dimeric, pentameric, orhexameric HIV binding molecule, e.g., an IgM antibody as providedherein.

In certain aspects, this disclosure provides a method for testing theability of a given binding molecule to bind to and effect killing of HIVreservoir cells. The method includes providing cells, e.g., cells fromchronically infected HIV patients, or a series of recombinant cell linesthat express an HIV antigen, e.g., an HIV protein, e.g., to an epitopeon the HIV spike protein, e.g., gp120 and/or gp41 at a series ofpredetermined levels from high copy number down to low, or even a singlecopy number. The cells can then be contacted with a dimeric, pentameric,or hexameric HIV binding molecule, e.g., an IgM antibody as providedherein under conditions that would allow antibody-dependent, T-celldependent, or complement-dependent killing of the cells, and recoveringthose binding molecules that can effect killing of the cells expressingthe lowest copy numbers of the HIV antigen. In certain aspects thedimeric, pentameric, or hexameric HIV binding molecule can directkilling of cells expressing a lower copy number of the HIV antigen thancells killed by an equivalent amount of a corresponding reference singlebinding unit molecule, e.g., a monospecific, bivalent IgG antibody orfragment thereof.

For instance, methods include screening of various binding moleculeswhose affinities and/or avidities for enveloped HIV viral particles of adifferent type, group, or clade, have not been determined. The presentmethods can be employed to identify more broadly neutralizing bindingmolecules that bind to the surface of HIV viral particles, on thesurface if HIV-infected cells, such as reservoir cells, or a combinationthereof. In this manner, additional binding molecules useful in themethods of the present disclosure can be identified and utilized, asdisclosed herein.

This screening method can be accomplished by contacting a test bindingmolecule known to specifically bind to an HIV or HIV-infected cell of afirst type, group, or clade with an HIV or HIV-infected cell of a secondtype, group, or clade, and measuring the affinity and/or avidity of thetest binding molecule for binding to the second HIV or infected cell.The dimeric, pentameric, or hexameric HIV binding molecule can thus betested for cross-reactivity that might not have been evident for asingle binding unit molecule having the same antigen binding domains.

The cells in such methods can be any cell capable of being infected byHIV, such as a human cell.

In certain aspects, this disclosure provides a method for directing morebroadly cross-reacting neutralization of HIV, or killing of HIVreservoir cells, where the method includes contacting an HIV virion, oran HIV-infected cell with a dimeric, pentameric, or hexameric HIVbinding molecule, e.g., an IgM antibody as described herein, where thevirus is of a different type, group, or clade than that typically boundby the one or more antigen binding domains of the binding molecule,where the binding molecule can direct virus neutralization, or killingof HIV reservoir cells of the different type, group, or clade, at ahigher potency than an equivalent amount of a corresponding referencesingle binding unit molecule, e.g., a monospecific, bivalent IgGantibody or fragment thereof. In certain aspects a dimeric, pentameric,or hexameric HIV binding molecule, e.g., an IgM antibody as providedherein can direct virus neutralization of more HIV types, subtypes orclades than an equivalent amount of a corresponding reference singlebinding unit molecule, where the corresponding reference single bindingunit molecule is, or comprises similar or identical VH and VL regions asat least one binding unit of a dimeric, pentameric, or hexameric HIVbinding molecule, e.g., an IgM antibody as provided herein.

For instance, methods include screening of various binding moleculeswhose affinities and/or avidities for enveloped HIV viral particles of adifferent type, group, or clade, have not been determined. The presentmethods can be employed to identify more broadly neutralizing bindingmolecules that bind to the surface of HIV viral particles, on thesurface if HIV-infected cells, such as reservoir cells, or a combinationthereof. In this manner, additional binding molecules useful in themethods of the present disclosure can be identified and utilized, asdisclosed herein.

This screening method can be accomplished by contacting a test bindingmolecule known to specifically bind to an HIV or HIV-infected cell of afirst type, group, or clade with an HIV or HIV-infected cell of a secondtype, group, or clade, and measuring the affinity and/or avidity of thetest binding molecule for binding to the second HIV or infected cell.The dimeric, pentameric, or hexameric HIV binding molecule can thus betested for cross-reactivity that might not have been evident for asingle binding unit molecule having the same antigen binding domains.

The cells in such methods can be any cell capable of being infected byHIV, such as a human cell.

Dimeric, pentameric, or hexameric HIV binding molecules for use in themethods provided herein can possess advantageous structural orfunctional properties compared to other binding molecules. For example,a dimeric, pentameric, or hexameric HIV binding molecule, e.g., an IgMantibody for use in the methods provided herein can possess improvedbinding characteristics in a biological assay, as described above,either in vitro or in vivo, than a corresponding reference singlebinding unit molecule, e.g., IgG or a variant, analog, or derivativethereof, as also described above. Biological assays include, but are notlimited to, T-cell Dependent Cell-mediated Cytotoxicity assays (TDCC),Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) assays,Complement-Dependent Cytotoxicity (CDC) assays, Cell-To-Cell Spread(CTCS) assays, complement-dependent virolysis assays, virusneutralization assays, cell attachment assays, viral egress assays,immunohistochemical assays, or direct cytotoxicity assays.

Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering a multimeric, e.g., a dimeric,pentameric, or hexameric HIV binding molecule, e.g., an IgM antibody asprovided herein to a subject in need thereof are well known to or arereadily determined by those skilled in the art in view of thisdisclosure. The route of administration of a multimeric binding moleculecan be, for example, oral, parenteral, by inhalation or topical. Theterm parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, orvaginal administration. While these forms of administration arecontemplated as suitable forms, another example of a form foradministration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. A suitablepharmaceutical composition can comprise a buffer (e.g. acetate,phosphate or citrate buffer), a surfactant (e.g. polysorbate), and insome embodiments a stabilizer agent (e.g. human albumin).

A dimeric, pentameric, or hexameric HIV binding molecule as providedherein can be administered in a pharmaceutically effective amount forthe in vivo treatment of diseases or disorders in which it is desirableto clear, remove or otherwise eliminate an HIV infection in a subjectinfected with HIV. In this regard, it will be appreciated that thedisclosed multimeric binding molecules can be formulated so as tofacilitate administration and promote stability of the active agent.Pharmaceutical compositions accordingly can comprise a pharmaceuticallyacceptable, non-toxic, sterile carrier such as physiological saline,non-toxic buffers, preservatives and the like. A pharmaceuticallyeffective amount of a dimeric, pentameric, or hexameric HIV bindingmolecule as provided herein means an amount sufficient to achieveeffective binding to a target and to achieve a therapeutic benefit.Suitable formulations are described in Remington's PharmaceuticalSciences (Mack Publishing Co.) 16th ed. (1980).

Certain pharmaceutical compositions provided herein can be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also can be administered by nasal aerosol or inhalation.Such compositions can be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of a dimeric, pentameric, or hexameric HIV binding moleculethat can be combined with carrier materials to produce a single dosageform will vary depending, e.g., upon the subject treated and theparticular mode of administration. The composition can be administeredas a single dose, multiple doses or over an established period of timein an infusion. Dosage regimens also can be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, a dimeric,pentameric, or hexameric HIV binding molecule as provided herein can beadministered to a subject in need of therapy in an amount sufficient toproduce a therapeutic effect. A multimeric binding molecule as providedherein can be administered to the subject in a conventional dosage formprepared by combining the antibody or antigen binding fragment, variant,or derivative thereof of the disclosure with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. The form and character of the pharmaceutically acceptablecarrier or diluent can be dictated by the amount of active ingredientwith which it is to be combined, the route of administration and otherwell-known variables.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of a dimeric, pentameric, or hexameric HIV bindingmolecule, that when administered brings about a positive therapeuticresponse with respect to treatment of a patient with a disease orcondition to be treated.

Therapeutically effective doses of the compositions disclosed herein,for treatment of HIV infection is desired, can vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. In certain aspects, the subject or patientis a human, but non-human mammals including transgenic mammals can alsobe treated. Treatment dosages can be titrated using routine methodsknown to those of skill in the art to optimize safety and efficacy.

The amount of a dimeric, pentameric, or hexameric HIV binding molecule,e.g., an IgM antibody to be administered is readily determined by one ofordinary skill in the art without undue experimentation given thisdisclosure. Factors influencing the mode of administration and therespective amount of a multimeric binding molecule include, but are notlimited to, the severity of the disease, the history of the disease, andthe age, height, weight, health, and physical condition of theindividual undergoing therapy. Similarly, the amount of a dimeric,pentameric, or hexameric HIV binding molecule to be administered will bedependent upon the mode of administration and whether the subject willundergo a single dose or multiple doses of this agent.

This disclosure also provides for the use of a dimeric, pentameric, orhexameric HIV binding molecule in the manufacture of a medicament fortreating, preventing, or managing a disease or disorder caused by HIVinfection.

This disclosure employs, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. (See, for example, Sambrook et al., ed. (1989) MolecularCloning A Laboratory Manual (2nd ed.; Cold Spring Harbor LaboratoryPress); Sambrook et al., ed. (1992) Molecular Cloning: A LaboratoryManual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985)DNA Cloning, Volumes I and II; Gait, ed. (1984) OligonucleotideSynthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins,eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984)Transcription And Translation; Freshney (1987) Culture Of Animal Cells(Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986);Perbal (1984) A Practical Guide To Molecular Cloning; the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Caloseds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold SpringHarbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In CellAnd Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described can be followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J. Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties. Thefollowing examples are offered by way of illustration and not by way oflimitation.

EXAMPLES Example 1: Construction and Assembly of Engineered Anti-HIVBinding Molecules

VH and VL regions of various HIV antibodies provided herein can becloned into IgG and IgM backgrounds by standard methods through acommercial contractor. The mature proteins presented below can beexpressed with a signal peptide to promote secretion.

HIV02:

The VH and VL of a human antibody specific for the CD4 binding site ongp120 provided in U.S. Pat. No. 8,637,036, presented herein as SEQ IDNO: 7 and SEQ ID NO: 8, respectively, were cloned into appropriatevectors to encode the human IgG and IgM heavy chains comprising theamino acid sequences SEQ ID NO: 113 and SEQ ID NO: 114, respectively,and the kappa light chain comprising SEQ ID NO: 115. The vectors weretransfected in to HEK293 cells (with, where appropriate, a vectorencoding a human wild-type or modified J-chain as described below) andexpression was permitted, producing the IgG molecule HIV02 IgG (HIV02G), the IgM molecule HIV02 IgM (HIV02M), the IgM+J (HIV02M+J), or theIgM+J containing a modified J-chain.

SEQ ID NO: 113: HIV02 Gamma 1 heavy chainQVQLVQSGGQMKKPGESMRISCQASGYEFIDCTLNWVRLAPGRRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTADDTAVYYCTRGKNCDYNWDFEHWGRGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGSEQ ID NO: 114: HIV02 Mu heavy chainQVQLVQSGGQMKKPGESMRISCQASGYEFIDCTLNWVRLAPGRRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTADDTAVYYCTRGKNCDYNWDFEHWGRGTPVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCYSEQ ID NO: 115 HIV02 Kappa light chainEIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTIRNLESGDFGLYYCQQYEFFGQGTKVQVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

HIV12:

The VH and VL of a human antibody specific for the V3/glycan region ongp120 provided in Buchacher, A., I., AIDS Res. Hum. Retroviruses10:359-369 (1994) and in WO 2011/035205, presented herein as SEQ ID NO:95 and SEQ ID NO: 96, respectively, were cloned into appropriate vectorsto encode the human IgG and IgM heavy chains comprising the amino acidsequences SEQ ID NO: 116 and SEQ ID NO: 117, respectively, and the kappalight chain comprising SEQ ID NO: 118. The vectors were transfected into HEK293 cells (with, where appropriate, a vector encoding a humanwild-type or modified J-chain as described below) and expression waspermitted, producing the IgG molecule HIV12 IgG (HIV12 G), the IgMmolecule HIV12 IgM (HIV12M), the IgM+J (HIV12M+J), or the IgM+Jcontaining a modified J-chain.

SEQ ID NO: 116: HIV12 Gamma 1 heavy chainEVQLVESGGGLVKAGGSLILSCGVSNFRISAHTMNWVRRVPGGGLEWVASISTSSTYRDYADAVKGRFTVSRDDLEDFVYLQMHKMRVEDTAIYYCARKGSDRLSDNDPFDAWGPGTVVTVSPASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGSEQ ID NO: 117: HIV12 Mu heavy chainEVQLVESGGGLVKAGGSLILSCGVSNFRISAHTMNWVRRVPGGGLEWVASISTSSTYRDYADAVKGRFTVSRDDLEDFVYLQMHKMRVEDTAIYYCARKGSDRLSDNDPFDAWGPGTVVTVSPASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGSEQ ID NO: 118: HIV12 Light chainDIQMTQSPSTLSASVGDTITITCRASQSIETWLAWYQQKPGKAPKLLIYKASTLKTGVPSRFSGSGSGTEFTLTISGLQFDDFATYHCQHYAGYSATFGQGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

HIV32:

The VH and VL of a human antibody specific for the CD4 binding site ongp120 provided in Moore, J. P., and J. Sodroski J. Virol 70:1863-1872(1996) and in WO 2006/044410, presented herein as SEQ ID NO: 99 and SEQID NO: 100, respectively, were cloned into appropriate vectors to encodethe human IgG and IgM heavy chains comprising the amino acid sequencesSEQ ID NO: 119 and SEQ ID NO: 120, respectively, and the kappa lightchain comprising SEQ ID NO: 121. The vectors were transfected in toHEK293 cells (with, where appropriate, a vector encoding a humanwild-type or modified J-chain as described below) and expression waspermitted, producing the IgG molecule HIV32 IgG (HIV32 G), the IgMmolecule HIV32 IgM (HIV32M), the IgM+J (HIV32M+J), or the IgM+Jcontaining a modified J-chain.

SEQ ID NO: 119: HIV32 Gamma 1 heavy chainQVQLQESGPGLVKPSQTLSLSCTVSGGSSSSGAHYWSWIRQYPGKGLEWIGYIHYSGNTYYNPSLKSRITISQHTSENQFSLKLNSVTVADTAVYYCARGTRLRTLRNAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGSEQ ID NO: 120: HIV32 Mu heavy chainQVQLQESGPGLVKPSQTLSLSCTVSGGSSSSGAHYWSWIRQYPGKGLEWIGYIHYSGNTYYNPSLKSRITISQHTSENQFSLKLNSVTVADTAVYYCARGTRLRTLRNAFDIWGQGTMVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCYSEQ ID NO: 121: HIV32 Kappa light chainQSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQHHPGKAPKLIISEVNNRPSGVPDRFSGSKSGNTASLTVSGLQAEDEAEYYCSSYTDIHNFVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

HIV72:

The VH and VL of a human antibody specific for the immunodominant regionof gp41, provided in Pincus S H, et al., J Immunol 170: 2236-2241(2003), presented herein as SEQ ID NO: 97 and SEQ ID NO: 98,respectively, were cloned into appropriate vectors to encode the humanIgG and IgM heavy chains comprising the amino acid sequences SEQ ID NO:122 and SEQ ID NO: 123, respectively, and the kappa light chaincomprising SEQ ID NO: 124. The vectors were transfected in to HEK293cells (with, where appropriate, a vector encoding a human wild-type ormodified J-chain as described below) and expression was permitted,producing the IgG molecule HIV72 IgG (HIV72 G), the IgM molecule HIV72IgM (HIV72M), the IgM+J (HIV72M+J), or the IgM+J containing a modifiedJ-chain.

SEQ ID NO: 122: HIV72 Gamma-1 heavy chainQVQLVQSGGGVFKPGGSLRLSCEASGFTFTEYYMTWVRQAPGKGLEWLAYISKNGEYSKYSPSSNGRFTISRDNAKNSVFLQLDRLSADDTAVYYCARADGLTYFSELLQYIFDLWGQGARVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGSEQ ID NO: 123: HIV72 Mu heavy chainQVQLVQSGGGVFKPGGSLRLSCEASGFTFTEYYMTWVRQAPGKGLEWLAYISKNGEYSKYSPSSNGRFTISRDNAKNSVFLQLDRLSADDTAVYYCARADGLTYFSELLQYIFDLWGQGARVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCYSEQ ID NO: 124 HIV72 Kappa Light chainDIVMTQSPDSLAVSPGERATIHCKSSQTLLYSSNNRHSIAWYQQRPGQPPKLLLYWASMRLSGVPDRFSGSGSGTDFTLTINNLQAEDVAIYYCHQYSSHPPTFGHGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Wild Type and Modified J-Chains:

Modified J-chains, V5J (SEQ ID NO: 108) and V10J (SEQ ID NO: 109),comprising an anti-CD3 ScFv corresponding to visilizumab fused to theN-terminus of the mature human J-chain via a 5-amino acid linker (GGGGS,SEQ ID NO: 101) or a 10-amino acid linker (GGGGSGGGGS, SEQ ID NO: 102),respectively were constructed by standard methods. See, e.g., PCTPublication No. WO 2015/153912.

The mature expressed construct has a molecular weight of about 45 kD andcan bind to soluble epsilon chain of CD3 (Sino Biological), or T-cells(data not shown).

The DNA constructs corresponding to the various anti-HIV heavy and lightchains as well as those corresponding to either the wild-type (wt)J-chain or a modified J-chain (e.g., V5J or V10J) sequences wereco-transfected into HEK293 cells, and proteins were expressed andpurified according to standard methods. HEK293 cells transfected withIgG or IgM+J versions of HIV02, HIV12, HIV32 and HIV72 antibodiesproduced sufficient protein to allow purification by standard methods.

Non-Reducing SDS-Native-PAGE.

Protein samples were loaded into a NativePAGE 3-12% Bis-Tris gel(Novex). Tris-Acetate SDS Running Buffer (Novex) was added and the gelwas run at 40V for 15 min and then at 90V for 2 hours. The gel was thenfixed in 40% methanol, 10% acetic acid for 10 minutes, stained using aColloidal Blue Staining Kit (Novex) for at least 3 hours andsubsequently de-stained in water.

Western Blot Detection.

After was complete, the gel was removed from the XCell SureLockMini-Cell and transferred to a PVDF membrane at 30 volts for 1 hour(refer to Life Technologies' manual). The PVDF membrane was then blockedwith 20 ml 3% BSA in PBST at 25° C. for 1 hour.

For anti-J-chain Western blots, anti-human J chain antibody (SP105,Thermo Fisher) was added at a 1:500 dilution in 3% bovine serum albumin(BSA) in phosphate-buffered saline, 0.05% TWEEN™ 20 (PBST) overnight at4° C. After washing with PBST four times at room temperature,horseradish peroxidase (HRP)-conjugated goat anti rabbit IgG (JacksonImmunology) was added at 1:5,000 dilution in 3% BSA in PBST and wasincubated for 1 hour at room temperature. The membrane was washed withPBST 4 times at room temperature and was developed by addition of 10 mlof HRP chemiluminescent substrate (Thermo Fisher) for 10 minutes beforeexposing the blot to film. Anti-J-chain antibody only reacts with IgMwhich is co-expressed with either unmodified J-chain or modifiedJ-chain.

Antibody Expression and Assembly:

Antibodies present in cell supernatants were recovered by affinitychromatography using CaptureSelectM (BAC, ThermoFisher catalog 2890.05)for IgM antibodies or Protein A for IgG antibodies per themanufacturer's instructions. The purified proteins were evaluated asoutlined below.

Expression and assembly of the HIV02 antibodies, as assessed bynon-reducing SDS native-PAGE is shown in FIG. 2A. HIV02 G expressed welland efficiently assembled into an IgG antibody. HIV02M expressed withouta J-chain produced a mixture of assembled high molecular weight IgM andlower molecular weight antibody forms. When HIV02M was expressed with awild-type J-chain (HIV02M+J), most of the material produced ran as afully assembled IgM antibody. HIV02M also properly assembled into a highmolecular weight bispecific IgM antibody when expressed with a modifiedJ-chain targeting CD3 (e.g., HIV02M+V10J; see FIG. 9).

Expression and assembly of HIV12M+J, HIV32M+J, and HIV72M+J is shown inFIG. 2B. All three IgM+J anti-HIV proteins properly assembled into highmolecular weight IgM antibodies, as evidenced by Western analysisdemonstrating the presence of J-chain in each of the proteins.

Expression and assembly of HIV72 G, HIV72M, HIV72M+J and HIV72M+V5J isshown in FIG. 2C. The heavy and light chains of HIV72 G expressed welland properly assembled into and IgG antibody (lane 2). The mu and lightchains of HIV72M, expressed without a J-chain, were also produced welland mostly assembled into a high molecular weight IgM antibody (lane 3).Co-expression of the HIV72 mu and light chains with wild type J-chain ora modified J-chain targeting CD3 (V5J) resulted in efficient assembly ofmonospecific (HIV72M+J; lane 4) or bispecific (HIV72M+V5J; lane 5) IgMantibodies, respectively, as evidenced by Coomassie staining of the gel(left) or by an anti-J-chain Western of the electrophoresed proteins.

Other Modified J-Chains:

Alternatively, modified J-chains can be constructed that allow bindingto the CD16 antigen on natural killer cells (NK cells). For example, amodified J-chain can be constructed that expresses a camelid VHH bindingdomain specific for CD16 (e.g., SEQ ID NO: 112). The binding domain islinked to a J-chain using a flexible amino acid linker (e.g., 15 aminoacids) to produce anti-CD16 camelid domain linked to the J chain (C15J).The bispecific IgM is expressed and purified as described above andassembly is confirmed by analyzing on non-reducing SDS-Native-PAGE gels.Further, incorporation of the modified J-chain (C15J, SEQ ID NO: 125)into the pentameric IgM is confirmed using the Western blot method.

SEQ ID NO: 125 EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

As described above, an expression vector containing DNA corresponding tothis sequence is synthesized and transfected into HEK293 cells alongwith the heavy and light chains for an anti-HIV gp120/41 IgM (SEQ ID NOs114 and 115) to produce protein which is then purified using the camelidantibody affinity matrix specific for IgM. J-chains fused to the newanti-CD16 binding domain with the 15 amino acid linker are able toincorporate into the IgM and the pentameric form of bi-specific IgM withthe corresponding J-chain is distinguishable from the hexameric formwithout a J-chain.

Example 2: Binding of HIV Specific IgM Binding Molecules to HIV Antigensand Antigen-Expressing Cells

HIV02 G and HIV02M+J were evaluated for binding to HIV gp120 by an ELISAassay. The assays were carried out by the following method. Wells of96-well microtiter plates (polystyrene, MaxiSorp ELISA plates, Nunc)were coated with gp120 antigen (strain HXB2, Immune Tech, cat#IT-001-0022p-PBS) at 1.0 μg/mL (high-density antigen) or dilutions downto 0.1 μg/mL (low density antigen) in 100 μL coating buffer (100 mMbicarbonate, pH 9.5) overnight at 4° C. The plates were then washed with0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, 100 μL ofthe serial diluted samples (purified protein or cell culturesupernatant) were added to the wells and incubated at room temperaturefor 1 hour. The plates were then washed and incubated with HRPconjugated mouse anti-human kappa antibody (Southern Biotech, 9230-05.1:6000 diluted in 2% BSA-PBS) for 1 hour. After 5 final washes using0.05% PBS-Tween, 100 μL TMB substrate (BD Biosciences, 555214) was addedto each well and incubated in the dark for 15 min. The reaction was thenstopped by adding 50 μL of 2N H2SO4 or 2N HCl per well. A450 data wasthen collected on a microplate reader and analyzed with Prism software(GraphPad) using a 4-parameter logistic model.

The results for binding to gp120 HBX2 are shown in FIG. 3A-F. At atypical high-antigen coating of 1 μg/mL (FIG. 3A), HIV02M+J exhibitedmore effective binding than the IgG version (HIV02 G), exhibiting anEC50 of 2,300 ng/mL vs 320 ng/mL, respectively, a 7-fold difference byweight. On a molar basis, HIV02M+J bound gp120 42-fold better than HIV02G at this coating concentration.

When the gp120 coating concentration was successively decreased from 1.0μg/mL to 0.8, 0.6, 0.4, 0.2 and 0.1 μg/mL (FIG. 3B to FIG. 3F), therebyreducing the antigen density on the plate, HIV02M+J continued to showstrong binding whereas HIV02 G binding decreased rapidly. At the lowestantigen density (FIG. 3F, 0.1 μg/mL coat), HIV02 G binding was barelydetectable.

Since binding of HIV02 G to gp120 fell off rapidly as the coatingconcentration was decreased, EC10 values were interpolated from thebinding curves to allow a binding comparison of antigen binding at allcoating concentrations. The results of this analysis are shown in FIG.4. As the gp120 coating concentration in the ELISA decreased from 1.0μg/mL to 0.1 μg/mL, the binding of IgM+J relative to that of the IgGincreased 5-fold to 20-fold on a weight basis. On a molar basis, theIgM+J bound gp120 120-fold better than the IgG when the antigen wascoated at the lowest concentration tested (0.1 μg/mL).

HIV02 G and HIV02M+J were further evaluated for binding to cellstransfected to express gp120 on their cell surface, by the followingmethod.

CHO cells engineered to express HBX2 gp140 (gp120+the extracellularportion of gp41; CHO-gp140) or gp160 (full-length protein; CHO-gp160) ontheir surface (Weiss et al., 1993 J Virol 67:7060-7066) were obtainedfrom the NIH AIDS Reagent Program (catalog #2284 & 2239) and culturedper their instructions. Cells were passaged in T75 flask at3×10{circumflex over ( )}6 cells per flask in 20 ml of media—cells werereplenished with fresh media containing 10 mM of Sodium Butyrate (Sigma,cat #B5887) 15 to 18 hrs prior to use. On the day of staining, the cellswere dislodged with 10 mL of cell dissociation buffer (Gibco, cat#131510). After aspirating off the media the cells were rinsed with PBSwithout Calcium & Magnesium. Ten mL of cell dissociation buffer wasadded to all the flasks and incubated for 20˜30 min at 37° C. The cellswere then dislodged by tapping the flask to create a single cellsuspension. An equal volume of the media was added to the flask toneutralize the cell dissociation buffer and live cell counts weredetermined using trypan blue exclusion on a cell counter (BioRad TC20).Cells at 1.5×10{circumflex over ( )}4/well in 60 μL of FACS 2% FBSbuffer (BD Pharmingen, cat #554656) were added to “V” bottom 96 wellplates. Antibodies and isotype controls were diluted in FACS 2% FBSbuffer and 50 μL were added to the respective wells such that the finalconcentration was 30 μg/mL. The plates were then incubated for 30 min at4° C. After washing the cells with 150 μL of FACS 2% FBS buffer, theplates were centrifuged (Sorvall Legend XIR centrifuge) at 1200 rpm for5 min and supernatants were gently aspirated without disturbing the cellpellets. Antibody binding was detected by incubating the treated cellswith AlexaFluor 647-labeled anti human kappa light chain (BioLegend, cat#316514) at 4° C. for 30 min. The wash step was repeated as above andthe cells were resuspended in 60 μL of FACS 2% FBS containing 1:1007_AAD (BD Pharm, cat #68981E). At least 1,000 events were acquired foreach sample on a FACSCALIBUR™ (Becton Dickinson) and data analysis wasdone in FLOWJO™ (FlowJo LLC).

The binding of HIV02 antibodies to CHO-gp140 cells is shown in FIG. 5Aand FIG. 5B. HIV02 G binding to CHO-gp140 cells was relatively low whenanalyzed by FACS (FIG. 5A). However, binding of the IgM version HIV02M+Jwas readily detected and much stronger (FIG. 5B).

Similarly, the binding of anti-HIV antibodies to cells chronically orlatently infected with HIV can be examined. Chronically infected celllines such as CEM cells (CEM-IIIb) (Popovic, M., et al., 1984. Science224:497-500) constitutively express HIV gp120/41 envelope glycoproteinon the cell surface whereas the latently infected cell lines ACH2 (FolksT M, et al., Proc Natl Acad Sci USA 86:2365-2368, 1989.), J1.1 (Perez VL, et al., J Immunol 147:3145-3148, 1991) and OM10 (Butera et al., 1991J Virol. 65(9):4645-4653) cell lines are activated with the cytokinetumor necrosis alpha to express HIV gp-120/41 envelope glycoprotein onthe cell surface. Serial dilutions of the monoclonal or bispecific IgMantibodies as described above as well as appropriate controls, e.g., IgGantibodies carrying equivalent anti-HIV binding domains, are incubatedwith the respective cell lines, washed to remove unbound antibody, thenmixed with a fluorochrome-labeled secondary antibody which is specificfor the isotype of antibody to be detected e.g., IgG, IgM, or the kappaor lambda light chain of an antibody. After incubation the binding ofmonoclonal or bispecific antibodies is analyzed by flow cytometry on aFACSCALIBUR™ (Becton Dickson).

Example 3: HIV Virus Neutralization by HIV Specific IgM BindingMolecules

In vitro HIV virus neutralization assays can be conducted using avariety of standard techniques, such as those described by Richman etal., PNAS USA, 100(7): 4144-4149, 2003.

In this assay, anti-HIV antibodies were examined for activity andpotency by using HIV pseudo-virus capable of a single round ofreplication. One or more HIV pseudo-viruses are produced byco-transfection of HEK293 cells with a sub-genomic plasmid,pHIV-1lucΔu3, that incorporates a firefly luciferase indicator gene anda second plasmid, pCXAS that expresses the HIV-1 envelope proteins ofinterest. Following transfection, pseudo-viruses were harvested andincubated for 1 hour at 37° C. with serial dilutions of the antibodiesto be tested. U87 cells that express CD4 plus the CCR5 and CXCR4co-receptors were then inoculated with the virus-antibody dilutions inthe absence of added cations. Virus infectivity was determined 72 hourpost-inoculation by measuring the amount of luciferase activityexpressed in infected cells. Neutralizing activity is described as thepercent inhibition of viral replication (luciferase activity) at eachantibody dilution compared with an antibody-negative control: %inhibition={1−[luciferase+Ab/luciferase−Ab]}×100.

The results of a virus neutralization assay are shown in FIG. 6.HIV02M+J was tested against a panel of viruses that included clades A,B, C, D, F, G, AE, AG, CRF07-BC and CRF08-BC. HIV02M+J successfullyneutralized all the viruses tested, exhibiting 75% to 100%neutralization at 10 μg/mL, indicating that HIV02M+J binds to all thedifferent gp120 proteins expressed by these clinically relevant HIVclades. Irrelevant isotype control IgM+J antibodies did not neutralizedany of the pseudo-viruses tested (data not shown).

Example 4: T-Cell Activation by HIV Specific IgM Binding Molecules

To demonstrate whether a bispecific HIV×CD3 antibody can activate Tcells upon binding to antigen-positive target cells, the following assaywas performed. Engineered Jurkat T cells (Promega CS176403) andCHO-gp140 cells expressing gp120/gp41 were cultured in RPMI (Invitrogen)supplemented with 10% Fetal Bovine Serum (Invitrogen). Serial dilutionsof HIV02M, HIV02M+J, and HIV02M+V10J were incubated with theantigen-expressing cells in 20 μL in a white 384 well assay plate for 2h at 37° C. with 5% CO₂. The engineered Jurkat cells (25000) were thenadded to the mixture to a final volume of 40 μL. The mixture wasincubated for 5 h at 37° C. with 5% CO₂. The cell mixtures were thenmixed with 20 μL lysis buffer containing luciferin (Promega, Cell TiterGlo) to allow measurement of luciferase reporter activity. Light outputwas measured by EnVision plate reader.

The results of an experiment examining the gp120-specific HIV02M,HIV02M+J, and HIV02M+V10J antibodies is shown in FIG. 7. Whereas themonospecific HIV02M and HIV02M+J antibodies were without activity, thegp120×CD3 bispecific HIV02M+V10J antibody caused T-cell activation in adose-dependent fashion.

PAGE analysis indicated that this preparation of HIV02M+V10J contained amixture of unassembled and fully assembled IgM proteins (FIG. 8, lanes 2& 8). The preparation was subsequently further purified bysize-exclusion chromatography utilizing a Waters 2695 HPLC systemequipped with a TOSOH G4000 SWXL chromatography column. The column wasequilibrated with degassed mobile phase (0.1 M Sodium Phosphate, 0.1 MSodium Sulfate pH 6.7) for 45 minutes at a flow rate of 1 ml/min and atemperature of 25° C. Samples were filtered and 100 μl was injected at aflow rate of 1 ml/min for a run time of 15 minutes. The absorbance wasmonitored at 280 nm using Waters EMPOWER™ software suite, which was alsoused to generate and analyze the chromatograms. One mL fractions werecollected using a Waters Fraction Collector II. Collected fractions weresubsequently concentrated and buffer exchanged into 20 mM citric acid,150 mM sodium chloride, pH 6.0 using Spin X UF-6 Concentrators (Corning,Ref #431486).

Analysis of the HIV02M+V10J antibody is shown in FIG. 8. Based onnon-reduced SDS-Native-PAGE, size-exclusion HPLC separated thepreparation into purified high molecular weight properly assembled IgMHIV02M+V10J bispecific (fractions 3 and 4) and unassembled, lowermolecular weight material (fractions 5 and 6).

The antigen-dependent T-cell activation induced by the highly purifiedHIV02M+V10J is shown in FIG. 9. In this assay, the purified HIV02M+V10Jantibody was more potent than the HIV02M+V10J starting material asevidenced by the higher activation signal.

Example 5: Complement-Dependent Cytotoxicity of HIV Specific IgM BindingMolecules

Antibodies of the IgM phenotype are particularly well-suited to use theefficient engagement of complement protein C1q to affect complementdependent cytotoxicity (CDC) activity on target cells. To measure CDC,recombinant cells expressing gp120 on their surface are used (e.g.,CHO-gp120, CHO-gp140, Jurkat-522 F/Y cells, or mammalian cellsexpressing membrane anchored trimeric forms of gp140, e.g., strain JR-FL(Go et al. 2015 J Virol 89:8245-8257)). The target cells are washed andresuspended in CDC assay medium (RPMI 1640, 10% heat-inactivated FBS) ata density of 1.0×10⁶ cells/mL and 10 μL/well is added to a Nunc 384-welltissue culture-treated white polystyrene plate. Serial 3-fold dilutionsof test antibodies including, e.g., a pentameric or hexameric HIVbinding molecule, e.g., an IgM antibody such as HIV02M, HIV02M+J, orHIV02M+V10J and appropriate controls, are prepared in assay medium, 10μL/well is added to the assay plate, and the plate is incubated for 2 hrat 37° C. in a 5% CO₂ incubator to allow opsonization to occur. Normalhuman serum complement (Quidel) is diluted to 30% in assay medium, and10 μL/well is added to the assay plate. The plate is incubated for 4 hrat 37° C. in a 5% CO₂ incubator. Cell Titer-Glo reagent (Promega) isthawed for use and 15 μL/well is added to the assay plate. The plate isgently mixed for 2 min on a plate shaker to lyse the cells and then foranother 10 min at room temperature before measuring luminescence on anEnVision plate reader (Perkin-Elmer). After subtracting backgroundsignal, percent viability is plotted against antibody concentration andEC50 values are determined using GraphPad Prism.

Alternatively, chronically HIV-infected cell lines (e.g., CEM-IIIb) orlatently infected cell lines (e.g., ACH2, J1.1, OM10) can be used. TheCEM-IIIb cells constitutively express the gp120/41 HIV envelopeglycoprotein on their cell surface, whereas the latent cell lines areactivated to express the gp120/41 HIV envelope glycoprotein that isindicative of HIV latency. Cells are seeded at 50,000 cells per well ina 96-well plate, serially diluted monoclonal or bispecific antibodiessuch as those described herein are added, and then human serumcomplement (Quidel cat. #A113) is added to a final concentration of 10percent of normal serum. The reaction mixture is incubated at 37° C. for4 hours. Cell Titer Glo reagent (Promega cat. #G7572) is added at avolume equal to the volume of culture medium present in each well. Theplate is shaken for 2 minutes, incubated for 10 minutes at roomtemperature, and luminescence is then measured on a luminometer.

Example 6: T-Cell Directed Killing of HIV Antigen-Expressing Cells byHIV Specific IgM Binding Molecules

Bispecific HIV×CD3 antibodies such as, but not limited to HIV02M+V10J,are tested for the induction of T-cell dependent cell cytotoxicity(TDCC) using HIV+ cells as targets. The target HIV+ cells used are cellsrecombinantly expressing gp120/gp41 on their surface (e.g., CHO-gp140,CHO-gp160, Jurkat-522 F/Y, or mammalian cells expressing membraneanchored trimeric forms of gp140, e.g., strain JR-FL (Go et al. 2015 JVirol 89:8245-8257)), chronically infected CEM cells (e.g., CEM-IIIb) orlatently infected cell lines (e.g., ACH2, J1.1, OM10). BispecificHIV×CD3 antibodies are serially diluted and mixed with the target cellsand PBMCs or purified/enriched CD8+ T cells from normal donors aseffector cells at various target:effector cell ratios. After 24-72 hoursthe amount of cell lysis or killing is analyzed by the addition of Celltiter glo (Promega) and luminescence is then measured on a luminometer.

Alternately, TDCC co-culture experiments can be conducted using CD8+T-cell acute lymphoblastic leukemia (TALL) cells. gp120/pg41-expressingcells (about 6×10³ cells), e.g., CHO-gp140, CHO-gp160, Jurkat-522 F/Y,or mammalian cells expressing membrane anchored trimeric forms of gp140,e.g., strain JR-FL (Go et al. 2015 J Virol 89:8245-8257)), chronicallyinfected CEM cells (e.g., CEM-IIIb) or latently infected cell lines(e.g., ACH2, J1.1, OM10), are co-cultured with 3×10⁴ TALL cells (ATCCCRL-11386) in the presence of different concentrations of testcompounds, e.g., HIV×CD3 bispecific antibodies, in 45 μL total volume ofRPMI 1640 media supplemented with 10% heat-inactivated FBS per well on a384-well black tissue culture plate. After 24 hours of incubation at 37°C. in a 5% CO₂ incubator, 15 μL of CytoTox-ONE substrate reagent(Promega, G7891) is added to each well to measure the level of LDHreleased from dead cells. The plates are shaken briefly to mix thereagents, and then incubated at room temperature for 90 min beforemeasuring fluorescence signal (485 nm for excitation and 615 nm foremission) on an EnVision plate reader (Perkin-Elmer). The data is thenanalyzed with Prism software (GraphPad) to determine the EC50.

Example 7: Treatment of HIV-Infected Animals with HIV IgM BindingMolecules

Effective control of HIV-1 infection in humans is achieved usingcombinations of antiretroviral therapy (ART) drugs (Bartlett et al.,AIDS 15(11): 1369-1377). However, when ART is stopped HIV virusre-appears or “rebounds” after a short period of time indicating a smallnumber of latently infected cells still remain in the ART-treatedindividual. This latent “reservoir” is the single biggest obstacle toHIV-infected individuals being cured of their infection. Therefore,since ART effectively reduces HIV infection to below detectable levelsthere is considerable interest in targeting and killing the reservoir oflatently infected cells which can result in the HIV-infected individualbeing cured of their infection.

A model of HIV latency has been described in humanized BLT mice (Dentonet al., J Virol. 2012 January; 86(1):630-4 (2012)) which has been usedby Horowitz et al., (Proc. Natl. Acad. Sci. USA. 2013 110(41):16538-43and Klein et al., Nature 492(7427): 118-122 (2012)) to analyze thecombination of ART plus immunotherapy to determine the possibility ofsuppressing or eliminating the rebound of virus after ART is stopped asa promising approach toward developing a cure for HIV infection.Humanized mice are screened at 8 weeks of age for reconstitution ofhuman lymphocytes as described by Klein et al. Nature 492(7427): 118-122(2012. Mice with measurable human lymphocytes are injectedintra-peritoneally with infectious HIV-1 YU2 virus and screened forviremia at 2 to 3 weeks post-infection by quantitativereverse-transcriptase PCR. Individual tablets of tenofovirdisproxil-fumarate (TDF; Gilead Sciences), emtricitabine (FTC; GileadSciences), raltegravir (RAL; Merck), and efavirenz (EFV; Bristol-MyersSquibb) are crushed into a fine powder form using a mortar and pestleand suspended in PBS. ART preparations are aliquotted into 200-μL dosesin sterile Eppendorf tubes and are administered daily by oral gavage at2.5, 1.5, 1.2, and 2.5 mg per mouse for TDF, FTC, RAL, and EFV,respectively, based on effective doses reported by Denton et al. (2012).Four (4) groups of humanized YU2-infected mice will begin receiving ARTby oral lavage daily on day 0 for 5 days and then groups 2, 3, and 4will begin receiving HIV02 G, HIV02M, and HIV02M+V10J, respectively, at20 mg/kg on day 5. Groups 2, 3, and 4 continue to receive antibodytherapy twice a week from day 5 to day 42. On day 21, ART is terminatedin all groups. HIV plasma viral load, cell-associated HIV DNA and RNAare determined at various time points through the duration of the studyto day 63 to determine the kinetics of virus rebound from latency.Similarly, other humanized mouse models can be used (see Xhang & Su 2012Cell. & Mol. Immunol. 9, 237-244).

Alternately, in vivo efficacy studies can be conducted in non-humanprimates. In one such model of chronic infection (Barouch et al. 2013Nature 503, 224-228), specific pathogen-free rhesus monkeys (Macacamulatta) that do not express the class I alleles Mamu-A*01, Mamu-B*08,and Mamu-B*17 associated with spontaneous virologic control are used.Groups are balanced for susceptible and resistant TRIM5α alleles. Groupsof 4 to 5 monkeys are randomly allocated to balance baseline viralloads. Animals are infected by the intrarectal route with rhesus-derivedSHIV-SF162P3 challenge stock for 9 months prior to antibodyadministration. Dimeric, pentameric, or hexameric HIV binding molecules,e.g., IgM antibodies such as HIV02M, HIV02M+J, or HIV02M+V10J, andappropriate controls, are administered to monkeys once or twice by theintravenous route at doses up to 10 mg/kg, and the monkeys are bled upto three times per week for assessment of viral loads. Alternatively,infected monkeys are treated with or without anti-retroviral therapy(e.g., ART) in addition to the dimeric, pentameric, or hexameric HIVbinding molecules. Once the anti-retroviral therapy has stopped, viralrebound is quantified. Similarly, infected monkeys can be treated withanti-retroviral therapy and, once stopped, then treated with thedimeric, pentameric, or hexameric HIV binding molecules and viralrebound is quantified.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. A multimeric binding molecule comprising two orfive bivalent binding units and a modified J-chain, wherein each bindingunit comprises two antibody heavy chain constant regions or fragmentsthereof, each associated with an antigen binding domain, wherein atleast one antigen binding domain specifically binds to a humanimmunodeficiency virus (HIV) antigen expressed on the surface of HIVviral particles, on the surface of HIV-infected cells, or a combinationthereof, and wherein the modified J-chain comprises a J-chain orfunctional fragment or variant thereof and a heterologous polypeptidedirectly or indirectly fused to the J-chain or variant or fragmentthereof.
 2. The binding molecule of claim 1, wherein the bindingmolecule is more potent than a reference single binding unit moleculecomprising the at least one antigen binding domain that specificallybinds to an HIV antigen, and wherein the reference single binding unitmolecule is an IgG antibody.
 3. The binding molecule of claim 1, whichis a dimeric binding molecule comprising two bivalent IgA binding units,wherein each binding unit comprises two IgA heavy chain constant regionsor fragments thereof each associated with an antigen binding domain, andwherein the IgA heavy chain constant regions each comprise a Cα3-tpdomain.
 4. The binding molecule of claim 1, which is a pentamericbinding molecule comprising five bivalent IgM binding units, whereineach binding unit comprises two human IgM heavy chain constant regionseach associated with an antigen binding domain, and wherein the IgMheavy chain constant regions each comprise a Cμ4-tp domain.
 5. Thebinding molecule of claim 1, wherein the heterologous polypeptide isfused to the N-terminus of the J-chain or fragment or variant thereof,the C-terminus of the J-chain or fragment or variant thereof, or to boththe N-terminus and C-terminus of the J-chain or fragment or variantthereof.
 6. The binding molecule of claim 1, wherein the heterologouspolypeptide comprises a binding domain, and wherein the binding domainof the heterologous polypeptide is an antibody or antigen bindingfragment thereof.
 7. The binding molecule of claim 6, wherein theantibody or fragment thereof comprises an Fab fragment, an Fab′fragment, an F(ab′)2 fragment, an Fd fragment, an Fv fragment, asingle-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment,a single domain antibody (VHH) or any combination thereof.
 8. Thebinding molecule of claim 7, wherein the antigen binding fragment is ascFv fragment or a VHH.
 9. The binding molecule of claim 6, wherein theheterologous polypeptide can specifically bind to a T-cell antigen, anNK cell antigen, a macrophage antigen, or a neutrophil antigen.
 10. Thebinding molecule of claim 9 wherein the heterologous polypeptide canspecifically bind to CD3 or CD8 on a T-cell, CD16, CD64, or NKG2D on anNK cell, CD14 on a macrophage, or CD16b or CD177 on a neutrophil. 11.The binding molecule of claim 9 wherein the heterologous polypeptide canspecifically bind to the T-cell antigen CD3 the NK cell antigen CD16.12. The binding molecule of claim 1, wherein each binding unit comprisestwo heavy chains each comprising a VH situated amino terminal to theconstant region or fragment thereof, and two immunoglobulin light chainseach comprising a VL situated amino terminal to an immunoglobulin lightchain constant region.
 13. The binding molecule of claim 12, wherein theat least one antigen binding domain specifically binds to the HIV spikeprotein.
 14. The binding molecule of claim 13, wherein at least onebinding unit comprises two antigen binding domains that specificallybind to an HIV antigen expressed on the surface of viral particles, onthe surface of HIV-infected cells, or a combination thereof, and whereinthe two heavy chains within the at least one binding unit are identical,and wherein the two light chains within the at least one binding unitare identical.
 15. The binding molecule of claim 13, comprising at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, or ten antigen binding domains thatspecifically bind to an HIV antigen expressed on the surface of viralparticles, on the surface of HIV-infected cells, or a combinationthereof.
 16. The binding molecule of claim 13, wherein the at least oneantigen binding domain comprises an antibody heavy chain variable region(VH) and an antibody light chain variable region (VL), wherein the VHand VL comprise the HCDR1, HCDR2, and HCDR3 regions, or HCDR1, HCDR2,and HCDR3 regions containing one or two single amino acid substitutions,and the LCDR1, LCDR2, and LCDR3 regions, or LCDR1, LCDR2, and LCDR3containing one or two single amino acid substitutions, of the VH and VLamino acid sequences of SEQ ID NO: 5 and SEQ ID NO:6, SEQ ID NO: 7 andSEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ IDNO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20,SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ IDNO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 andSEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ IDNO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46,SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ IDNO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 55and SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 andSEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ IDNO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO:68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72,SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 6, SEQ IDNO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81and SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 andSEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 and SEQ IDNO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO:94, SEQ ID NO: 95 and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, orSEQ ID NO: 99 and SEQ ID NO: 100, respectively; or wherein the at leastone antigen binding domain comprises an antibody heavy chain variableregion (VH) and an antibody light chain variable region (VL), whereinthe VH and VL comprise, respectively, amino acid sequences that are atleast 95% or 100% identical to amino acid sequences of SEQ ID NO: 5 andSEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14,SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ IDNO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ IDNO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40,SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ IDNO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 andSEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, SEQ ID NO: 57 and SEQ IDNO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO:62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66,SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ IDNO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75and SEQ ID NO: 6, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO:84, SEQ ID NO: 85 and SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88,SEQ ID NO: 89 and SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, SEQ IDNO: 93 and SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, SEQ ID NO: 97and SEQ ID NO: 98, or SEQ ID NO: 99 and SEQ ID NO:
 100. 17. An isolatedIgM antibody comprising a modified J-chain, and five binding units, eachcomprising two heavy chains and two light chains, wherein each heavychain or fragment thereof comprises a human Mu constant region orfragment thereof, and the heavy chain variable region amino acidsequence SEQ ID NO: 95, and wherein each light chain comprises a humankappa constant region and the light chain variable region amino acidsequence SEQ ID NO: 96; wherein the antibody or fragment thereof canassemble into a pentameric IgM antibody that can specifically bind tothe CD4 binding site of the HIV spike glycoprotein, and wherein themodified J-chain comprises a J-chain or functional fragment or variantthereof and an antigen binding scFv antibody fragment that binds to CD3directly or indirectly fused to the J-chain or variant or fragmentthereof.
 18. The IgM antibody of claim 17, wherein the modified J-chaincomprises SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO:111.
 19. A composition comprising the multimeric binding molecule ofclaim
 1. 20. A method of controlling or treating human immunodeficiencyvirus (HIV) infection, or controlling HIV infectivity, comprisingcontacting HIV virions, HIV-infected cells, or a mixture thereof withthe multimeric binding molecule of claim 1; wherein the binding moleculeis more potent in preventing, controlling or treating HIV infection, orin controlling HIV infectivity, than a corresponding IgG antibodycomprising an identical HIV-binding antigen binding domain.